Genetically engineered swine influenza virus and uses thereof

ABSTRACT

The present invention relates, in general, to attenuated swine influenza viruses having an impaired ability to antagonize the cellular interferon (IFN) response, and the use of such attenuated viruses in vaccine and pharmaceutical formulations. In particular, the invention relates to attenuated swine influenza viruses having modifications to a swine NS1 gene that diminish or eliminate the ability of the NS1 gene product to antagonize the cellular IFN response. These viruses replicate in vivo, but demonstrate decreased replication, virulence and increased attenuation, and therefore are well suited for use in live virus vaccines, and pharmaceutical formulations.

This application is a divisional of copending U.S. application Ser. No.13/304,175, filed Nov. 23, 2011, which is a divisional of U.S.application Ser. No. 11/628,292, filed Feb. 6, 2008, now U.S. Pat. No.8,124,101, which is the National Stage of International Application No.PCT/US2005/019382, filed Jun. 1, 2005, which claims the benefit under 35U.S.C. 119(e) of U.S. Provisional Application No. 60/576,418, filed Jun.1, 2004, each of which is incorporated by reference herein in itsentirety.

This invention was made with United States Government support underNational Institutes of Health grant number AI95357 to St. JudeChildren's Research Hospital. The United States Government has certainrights in this invention.

The present specification contains a Sequence Listing, which has beensubmitted in electronic format via EFS-Web and is hereby incorporated byreference in its entirety. The Sequence Listing is provided as acomputer readable format (CRF) file entitledSubstitute_SEQLIST_6923-241-999, which was created on Jun. 8, 2015, andis 20,749 bytes in size.

1. FIELD OF THE INVENTION

The present invention relates, in general, to attenuated swine influenzaviruses having an impaired ability to antagonize the cellular interferon(IFN) response, and the use of such attenuated viruses in vaccine andpharmaceutical formulations. In particular, the invention relates toattenuated swine influenza viruses having modifications to a swine NS1gene that diminish or eliminate the ability of the NS1 gene product toantagonize the cellular IFN response. These viruses replicate in vivo,but demonstrate decreased virulence and increased attenuation, andtherefore are well suited for use in live virus vaccines, andpharmaceutical formulations.

2. BACKGROUND 2.1 Influenza Virus

Virus families containing enveloped single-stranded RNA of thenegative-sense genome are classified into groups having non-segmentedgenomes (Paramyxoviridae, Rhabdoviridae, Filoviridae and Borna DiseaseVirus) or those having segmented genomes (Orthomyxoviridae, Bunyaviridaeand Arenaviridae). The Orthomyxoviridae family, described in detailbelow, and used in the examples herein, includes the viruses ofinfluenza, types A, B and C viruses, as well as Thogoto and Dhoriviruses and infectious salmon anemia virus.

The influenza virions consist of an internal ribonucleoprotein core (ahelical nucleocapsid) containing the single-stranded RNA genome, and anouter lipoprotein envelope lined inside by a matrix protein (M1). Thesegmented genome of influenza A virus consists of eight molecules (sevenfor influenza C) of linear, negative polarity, single-stranded RNAswhich encode eleven polypeptides, including: the RNA-dependent RNApolymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which formthe nucleocapsid; the matrix membrane proteins (M1, M2); two surfaceglycoproteins which project from the lipid containing envelope:hemagglutinin (HA) and neuraminidase (NA); the nonstructural protein(NS1), nuclear export protein (NEP); and the proapoptotic factor PB1-F2.Transcription and replication of the genome takes place in the nucleusand assembly occurs via budding on the plasma membrane. The viruses canreassort genes during mixed infections.

Influenza virus adsorbs via HA to sialyloligosaccharides in cellmembrane glycoproteins and glycolipids. Following endocytosis of thevirion, a conformational change in the HA molecule occurs within thecellular endosome which facilitates membrane fusion, thus triggeringuncoating. The nucleocapsid migrates to the nucleus where viral mRNA istranscribed. Viral mRNA is transcribed by a unique mechanism in whichviral endonuclease cleaves the capped 5′-terminus from cellularheterologous mRNAs which then serve as primers for transcription ofviral RNA templates by the viral transcriptase. Transcripts terminate atsites 15 to 22 bases from the ends of their templates, where oligo(U)sequences act as signals for the addition of poly(A) tracts. Of theeight viral RNA molecules so produced, six are monocistronic messagesthat are translated directly into the proteins representing HA, NA, NPand the viral polymerase proteins, PB2, PB1 and PA. The other twotranscripts undergo splicing, each yielding two mRNAs which aretranslated in different reading frames to produce M1, M2, NS1 and NEP.The PB1 segment encodes a second protein, the nonstructural PB1-F2protein, by using of an alternative ATG. In other words, the eight viralRNA segments code for eleven proteins: nine structural and twononstructural. A summary of the genes of the influenza virus and theirprotein products is shown in Table I below.

TABLE I INFLUENZA VIRUS GENOME RNA SEGMENTS AND CODING ASSIGNMENTS^(a)Length_(d) Length_(b) Encoded (Amino Molecules Segment (Nucleotides)Polypeptide_(c) Acids) Per Virion Comments 1 2341 PB2 759 30-60 RNAtranscriptase component; host cell RNA cap binding 2 2341 PB1 757 30-60RNA transcriptase component; initiation of transcription PB1-F2 87Proapoptotic factor 3 2233 PA 716 30-60 RNA transcriptase component 41778 HA 566 500 Hemagglutinin; trimer; envelope glycoprotein; mediatesattachment to cells 5 1565 NP 498 1000  Nucleoprotein; associated withRNA; structural component of RNA transcriptase 6 1413 NA 454 100Neuraminidase; tetramer; envelope glycoprotein 7 1027 M₁ 252 3000 Matrix protein; lines inside of envelope M₂ 96 ? Structural protein inplasma membrane; spliced mRNA 8 890 NS₁ 230 Nonstructural protein;function unknown NEP 121 ? Nuclear export protein; spliced mRNA^(a)Adapted from R. A. Lamb and P. W. Choppin (1983), Annual Review ofBiochemistry, Volume 52, 467-506. _(b)For A/PR/8/34 strain_(c)Determined by biochemical and genetic approaches _(d)Determined bynucleotide sequence analysis and protein sequencing

The pathogenicity of influenza viruses is modulated by multiple virusand host factors. Among the host factors that fight virus infections,the type I interferon (IFNα/β) system represents a powerful antiviralinnate defense mechanism which was established relatively early in theevolution of eukaryotic organisms (Garcia-Sastre, 2002, Microbes Infect4:647-55). The antiviral IFNα/β system involves three major steps: (i)detection of viral infection and IFNα/β secretion, (ii) binding ofIFNα/β to its receptors and transcriptional induction ofIFNα/β-stimulated genes, and (iii) synthesis of antiviral enzymes andproteins. Most viruses, however, have acquired specific geneticinformation encoding IFNα/β antagonist molecules, which effectivelyblock one or more steps of the antiviral IFNα/β system. Influenza Aviruses express a non-structural protein in infected cells, the NS1protein (described in detail, infra), which counteracts the cellularIFNα/β response (Garcia-Sastre et al., 1998, Virology 252:324-30).

The influenza A virus genome contains eight segments of single-strandedRNA of negative polarity, coding for two nonstructural and ninestructural proteins. The nonstructural protein NS1 is abundant ininfluenza virus infected cells, but has not been detected in virions.NS1 is a phosphoprotein found in the nucleus early during infection andalso in the cytoplasm at later times of the viral cycle (King et al.,1975, Virology 64: 378). Studies with temperature-sensitive (ts)influenza mutants carrying lesions in the NS gene suggested that the NS1protein is a transcriptional and post-transcriptional regulator ofmechanisms by which the virus is able to inhibit host cell geneexpression and to stimulate viral protein synthesis. Like many otherproteins that regulate post-transcriptional processes, the NS1 proteininteracts with specific RNA sequences and structures. The NS1 proteinhas been reported to bind to different RNA species including: vRNA,poly-A, U6 snRNA, 5′ untranslated region as of viral mRNAs and ds RNA(Qiu et al., 1995, RNA 1: 304; Qiu et al., 1994, J. Virol. 68: 2425;Hatada Fukuda 1992, J Gen Virol. 73:3325-9. Expression of the NS1protein from cDNA in transfected cells has been associated with severaleffects: inhibition of nucleo-cytoplasmic transport of mRNA, inhibitionof pre-mRNA splicing, inhibition of host mRNA polyadenylation andstimulation of translation of viral mRNA (Fortes, et al., 1994, EMBO J.13: 704; Enami et al., 1994, J. Virol. 68: 1432; de la Luna et al.,1995, J. Virol. 69:2427; Lu et al., 1994, Genes Dev. 8:1817; Park etal., 1995, J. Biol Chem. 270, 28433; Nemeroff et al., 1998, Mol. Cell.1:1991; Chen et al., 1994, EMBO J. 18:2273-83). In particular, the NS1protein has three domains that have been reported to have a number ofregulatory functions during influenza virus infection. Theamino-terminal 73 amino acids are responsible for binding to RNAs (Qianet al., 1995, RNA 1:948-956), particularly double stranded RNAs,conferring to the virus the ability to escape the interferon α/βresponse (Donelan et al., 2003, J. Virol. 77:13257-66). The centralportion of the protein interacts with the eukaryotic translationinitiation factor 4GI facilitating preferential translation of viralmRNAs over host mRNAs (Aragón et al., 2000, Mol. Cell Biol.20:6259-6268). The carboxy-terminus or the effector domain has beenshown to inhibit host mRNA processing, specifically, inhibition of hostmRNA polyadenylation (Nemeroff et al., 1998, Mol. Cell 1:991-1000),binding to poly(A) tails of mRNA inhibiting nuclear export (Qiu andKrug, 1994, J. Virol. 68:2425-2432) and inhibition of pre-mRNA splicing(Lu et al., 1994, Genes Dev. 8:1817-1828).

Studies of human recombinant influenza virus lacking the NS1 gene(delNS1) showed that this virus could only replicate in IFN-incompetentsystems such as STAT1−/− mice or Vero cells; thus the NS1 protein isresponsible for IFN antagonist activity (Garcia-Sastre et al., 1998,Virology 252:324-330). Also, it has been shown that human influenzaviruses with truncated NS1 proteins are attenuated in mice (Egorov etal., 1998, J. Virol. 72:6437-6441) and provide protection againstwild-type challenge (Talon et al., 2000, Proc. Natl. Acad. Sci. USA97:4309-4314).

2.2 Swine Influenza Virus

Swine influenza (SI) is an acute respiratory disease of swine caused bytype A influenza viruses. Its severity depends on many factors,including host age, virus strain, and secondary infections (Easterday,1980, Philos Trans R Soc Lond B Biol Sci 288:433-7). Influenza A virusesare segmented negative-strand RNA viruses and can be isolated from anumber of other animal host species, including birds, humans, horses,whales, and mink. Although whole influenza viruses rarely cross thespecies barrier, gene segments can cross this barrier through theprocess of genetic reassortment, or genetic shift. Since pigs supportthe replication of both human and avian influenza A viruses (Kida etal., 1994, J Gen Virol 75:2183-8), they have been postulated to play animportant role in interspecies transmission by acting as a “mixingvessel” for reassortment between viruses specific to different hostspecies (Scholtissek, 1994, Eur J Epidemiol 10:455-8). This may lead tothe generation of novel influenza viruses capable of crossing thespecies barrier to humans. There are three subtypes of SI viruses (SIV)currently circulating in pigs in the U.S.: H1N1, H3N2, and H1N2 (Olsen,2002, Virus Res 85:199-210; Karasin et al., 2002, J Clin Microbiol40:1073-9; Karasin et al., 2000, Virus Res 68:71-85; Olsen et al., 2000,Arch Virol 145:1399-419; Webby et al., 2000, J Virol 74:8243-51; Webbyet al., 2001, Philos Trans R Soc Lond B Biol Sci 356:1817-28; Zhou,2000, Vet Microbiol 74:47-58). Before 1998, mainly “classical” H1N1 SIVswere isolated from swine in the United States (Kida et al., 1994, J GenVirol 75:2183-8; Scholtissek, 1994, Eur J Epidemiol 10:455-8; Olsen etal., 2000, Arch Virol. 145:1399-419). In 1998, SIVs of the subtype H3N2were isolated in multiple states in the United States. These viruseswere generated by reassortment between human, avian and classical swineviruses, they are undergoing rapid evolution and in general they causemore severe disease than classical H1N1 SIV.

Pathogenicity of influenza viruses is modulated by multiple virus andhost factors. Among the host factors that fight virus infections, thetype I interferon (IFNα/β) system represents a powerful antiviral innatedefense mechanism which was established relatively early in theevolution of eukaryotic organisms (Garcia-Sastre, 2002, Microbes Infect4:647-55). The antiviral IFNα/β system involves three major steps: (i)detection of viral infection and IFNα/β secretion, (ii) binding ofIFNα/β to its receptors and transcriptional induction ofIFNα/β-stimulated genes, and (iii) synthesis of antiviral enzymes andproteins. Most viruses, however, have acquired specific geneticinformation encoding IFNα/β antagonist molecules, which effectivelyblock one or more steps of the antiviral IFNα/β system. Influenza Aviruses express a non-structural protein in infected cells, the NS1protein, which counteracts the cellular IFNα/β response (Garcia-Sastreet al., 1998, Virology 252:324-30).

Influenza infection in pigs was first reported in 1918 and the firstswine influenza viruses were isolated from pigs in 1930 (Shope, R. E.,1931, J. Exp. Med. 54:373-385). These first isolates were theprogenitors of what is recognized as the H1N1 lineage of swine influenzaA viruses. From 1930 to the 1990s, influenza in North American pigs wascaused almost exclusively by infection with H1N1 swine viruses. Adramatic shift in the pattern of swine influenza began around 1997, whenan unexpected and substantial increase in H3 seropositivity (8%) wasdetected, and H3N2 viruses began to be isolated from pigs in both the USand Canada (Olsen et al., 2000, Arch. Virol. 134:1399-1419).Furthermore, reassortment between H3N2 viruses and H1N1 swine virusesresulted in the detection of second generation H1N2 reassortant viruses(Karasin et al., 2000, J. Clin. Microbiol. 38:2453-2456; Karasin et al.,2002, J. Clin Microbiol. 40:1073-1079). In addition, avian H4N6 virusesof duck origin have been isolated from pigs in Canada (Karasin et al.,2000, J. Virol. 74:9322-9327). The generation of these novel viruses inaddition to the described antigenic drift variants of H1N1 swineinfluenza viruses, introduce potential veterinary and human publichealth implications.

In 1998, a new strain of swine influenza virus to which pigs had littleimmunity sickened every pig in an operation of 2400 animals. Althoughthere has been only one influenza subtype which has sickened NorthAmerican pigs since 1930, in the last few years a quick succession ofnew flu viruses has been sweeping through North America's 100 millionpigs. After years of stability, the North American swine flu virus hasjumped onto an evolutionary fast track, bringing out variants everyyear. This has had not only an undesired effect on the farming industryand a negative economic impact, but, there is also concern by expertsthat the evolving swine flu increases the likelihood that a novel viruswill arise that is transmissible among humans. Fortunately, the new pigstrains that have appeared in North America so far do not appear toreadily infect humans.

2.3 Attenuated Viruses

Inactivated virus vaccines are prepared by “killing” the viral pathogen,e.g., by heat or formalin treatment, so that it is not capable ofreplication. Inactivated vaccines have limited utility because they donot provide long lasting immunity and, therefore, afford limitedprotection. An alternative approach for producing virus vaccinesinvolves the use of attenuated live virus vaccines. Attenuated virusesare capable of replication but are not pathogenic, and, therefore,provide for longer lasting immunity and afford greater protection.However, the conventional methods for producing attenuated virusesinvolve the chance isolation of host range mutants, many of which aretemperature sensitive; e.g., the virus is passaged through unnaturalhosts, and progeny viruses which are immunogenic, yet not pathogenic,are selected.

A conventional substrate for isolating and growing influenza viruses forvaccine purposes are embryonated chicken eggs. Influenza viruses aretypically grown during 2-4 days at 37° C. in 10-12 day old eggs.Although most of the human primary isolates of influenza A and B virusesgrow better in the amniotic sac of the embryos, after 2 to 3 passagesthe viruses become adapted to grow in the cells of the allantoic cavity,which is accessible from the outside of the egg (Murphy, B. R., and R.G. Webster, 1996. Orthomyxoviruses pp. 1397-1445. In Fields Virology.Lippincott-Raven P. A.).

Recombinant DNA technology and genetic engineering techniques, intheory, would afford a superior approach to producing an attenuatedvirus since specific mutations could be deliberately engineered into theviral genome. However, the genetic alterations required for attenuationof viruses are not known or predictable. In general, the attempts to userecombinant DNA technology to engineer viral vaccines have mostly beendirected to the production of subunit vaccines which contain only theprotein subunits of the pathogen involved in the immune response,expressed in recombinant viral vectors such as vaccinia virus orbaculovirus. More recently, recombinant DNA techniques have beenutilized in an attempt to produce herpes virus deletion mutants orpolioviruses which mimic attenuated viruses found in nature or knownhost range mutants. Until 1990, the negative strand RNA viruses were notamenable to site-specific manipulation at all, and thus could not begenetically engineered.

Although these viruses are beneficial because they are immunogenic andnot pathogenic, they are difficult to propagate in conventionalsubstrates for the purposes of making vaccines. Furthermore, attenuatedviruses may possess virulence characteristics that are so mild as to notallow the host to mount an immune response sufficient to meet subsequentchallenges.

Human influenza viruses does not replicate efficiently in birds, andvice versa due to differences in receptors which bind the viruses. Incontrast, pigs are uniquely susceptible to infection with human andavian viruses because they possess receptor types present both in humansand avian influenza viruses. As a result, pigs have been hypothesized tobe the “mixing vessel” hosts for human-avian virus reassortment andthere is support for this theory from several studies. See, e.g., Shu etal., 1994, Virology 202:825-33; Scholtissek, 1990, Med. PrinciplesPract. 2:65-71; Zhou et al., 1999 J Virol. 73:8851-6. This mixingfacilitates the generation of novel human influenza virus strains andthe initiation of influenza pandemics.

Inactivated or “killed” influenza virus preparations are the onlyinfluenza vaccines currently licensed in the United States. Analternative approach for producing virus vaccines to the inactivatedvirus vaccines in which the viral pathogen is “killed”, involves the useof attenuated live virus vaccines which are capable of replication butare not pathogenic. Live vaccines which are administered intranasallymay have advantages over their inactivated counterparts. Firstly, livevaccines are thought to induce improved cross-reactive cell-mediatedcytotoxicity as well as a humoral antibody response, providing betterprotection than inactivated vaccines (Gorse and Belshe, 1990, J. Clin.Microbiol. 28:2539-2550; and Gorse et al., 1995, J. Infect. Dis.172:1-10). Secondly, live vaccines also have the advantage of intranasaladministration which avoids the swelling and muscle sorenessoccasionally associated with the intramuscular administration ofinactivated adjuvanted vaccines. These live vaccines have been reportedto induce not only humoral responses against homotypic influenza virusbut also crossreactive cell-mediated cytotoxicity. Further advantages oflive vaccines include the ease of intranasal administration, inductionof mucosal immunity, longer lasting immunity, and its costeffectiveness. These are all important considerations regardingpotential swine influenza vaccines.

Thus, new and more effective vaccines and immunogenic formulations forpreventing swine influenza virus infections generated by such technologyare needed.

3. SUMMARY OF THE INVENTION

The present invention provides attenuated swine influenza viruses havingan impaired ability to antagonize the cellular interferon (IFN)response, methods for producing such attenuated swine influenza viruses,and the use of such viruses in vaccine and pharmaceutical formulations.Such viruses are capable of generating an immune response and creatingimmunity but not causing illness or causing fewer and/or less severesymptoms, i.e., the viruses have decreased virulence. Therefore, theyare ideal candidates for live virus vaccines. Moreover, the attenuatedviruses can induce a robust IFN response which has other biologicalconsequences in vivo, affording protection against subsequent infectiousdiseases and/or inducing antitumor responses. Therefore, the attenuatedviruses can be used pharmaceutically, for the prevention or treatment ofother infectious diseases and/or IFN-treatable diseases.

The invention is based, in part, on the Applicants' discovery that swineinfluenza viruses engineered to contain or containing a deletion(s) inthe NS1 gene have impaired replication relative to wild-type swineinfluenza viruses as demonstrated by fewer lung lesions upon infectionof pigs, reduced viral titers in bronchoalveolar lavage fluid (BALF) andreduced detection of virus on nasal swabs. Surprisingly, and contrary toresults seen with human influenza virus in mouse models, the length ofthe NS1 protein does not correlate with the level of attenuation of theswine influenza virus. Applicants have discovered that with swineinfluenza, a mutant virus with the shortest NS1 protein is the leastattenuated. In other words, swine influenza viruses containing shorterdeletions in the NS1 gene exhibit a greater attenuation in vivo thanswine influenza viruses containing longer deletions in their NS1 gene,or wild type swine influenza viruses. Applicants have further discoveredthat the recombinant swine influenza viruses are impaired in theirability to inhibit IFN production in vitro, and they do not replicate asefficiently as the parental recombinant strain in 10-day old embryonatedhen eggs, in MDCK cells, in PK-15 cells or in an in vivo pig model.While not intending to be bound to any theory or explanation for themechanism of action of the swine influenza virus NS1 deletion mutants invivo, the attenuated features of such viruses are presumably due totheir levels of NS1 protein expression, their ability to induce a robustcellular IFN response, and their impaired ability to antagonize such aresponse. However, the beneficial features of such viruses may not besolely attributable to their effect on the cellular interferon response.Indeed, alterations in other activities associated with NS1, such as,alteration of pre-mRNA splicing, inhibition of cellular mRNApolyadenylation, poly(A)-containing mRNA nucleocytoplasmic transport,and stimulation of viral protein synthesis, may contribute to thedesired attenuated phenotype achieved by the introduction of mutationsin the NS1 gene of swine influenza virus.

An attenuated swine influenza virus of the present invention comprises amutation in a swine influenza NS1 gene that diminishes the ability ofthe NS1 gene product to antagonize the cellular interferon response. Inone embodiment, an attenuated swine influenza virus of the inventioncomprises a genome comprising a mutation in a swine influenza virus NS1gene that diminishes the ability of the NS1 gene product to antagonize acellular interferon response, and permits the attenuated virus, at amultiplicity of infection (MOI) of between 0.0005 and 0.001, 0.001 and0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001,0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5,5.0, 5.5, or 6.0, to grow to titers between approximately 1 toapproximately 100 fold, approximately 5 to approximately 80 fold,approximately 20 to approximately 80 fold, or approximately 40 toapproximately 80 fold, approximately 1 to approximately 10 fold,approximately 1 to approximately 5 fold, approximately 1 toapproximately 4 fold, approximately 1 to approximately 3 fold,approximately 1 to approximately 2 fold, approximately 3 toapproximately 15 fold, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100fold lower than wild-type swine influenza virus in cells (e.g., cells ofa human (e.g., PerC6, a producer cell line derived from human embryonicretinoblasts transformed with the E1 region of Adenovirus 5), mouse,chicken (e.g., chicken embryo fibroblasts), rat, birds, or pig (e.g.,PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells,LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 or SJPLcells)), as determined approximately 2 to 10 days, 3 to 7 days, 3 to 5days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 days post-infection when propagatedunder the same conditions. The titers of attenuated and wild-type swineinfluenza viruses can be determined utilizing any technique well-knownin the art or described herein, (e.g., hemagglutination assays, plaqueassays, tissue culture infectious dose 50 (TCID50), egg infectious dose50 (EID50), etc.) and the viruses can be propagated under conditionsdescribed herein or well-known in the art (e.g., in pig cells, MDCKcells (e.g., in MEM, 10% v/v fetal calf serum (FCS), 1%penicillin/streptomycin at 37° C. in a 5% CO₂ humidified incubator) orembryonated chicken eggs (e.g., in a stationary incubator at 37° C. with55% relative humidity). Alternatively, the viruses can be propagated incells (e.g., in pig cells, MDCK cells, etc.) that are grown inserum-free or serum reduced (e.g., TPCK trypsin) medium.

In a specific embodiment, an attenuated swine influenza virus of theinvention comprises a genome comprising a mutation in a swine influenzavirus NS1 gene that diminishes the ability of the NS1 gene product toantagonize a cellular interferon response, and permits the attenuatedvirus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001,0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007,0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 toapproximately 100 fold, approximately 5 to approximately 80 fold,approximately 20 to approximately 80 fold, or approximately 40 toapproximately 80 fold, approximately 1 to approximately 10 fold,approximately 1 to approximately 5 fold, approximately 1 toapproximately 4 fold, approximately 1 to approximately 3 fold,approximately 1 to approximately 2 fold, approximately 3 toapproximately 15 fold, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100fold lower than wild-type swine influenza virus in cells (e.g., cells ofa human (e.g., PerC6, a producer cell line derived from human embryonicretinoblasts transformed with the E1 region of Adenovirus 5), mouse,chicken (e.g., chicken embryo fibroblasts), rat, birds, or pig (e.g.,PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells,LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 or SJPLcells)), as determined approximately 2 to 10 days, 3 to 7 days, 3 to 5days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 days post-infection when propagatedunder the same conditions. The titers of attenuated and wild-type swineinfluenza viruses can be determined utilizing any technique well-knownin the art or described herein, (e.g., hemagglutination assays, plaqueassays, etc.) and the viruses can be propagated under conditionsdescribed herein or well-known in the art (e.g., in pig cells, MDCKcells (e.g., in MEM, 10% v/v fetal calf serum (FCS), 1%penicillin/streptomycin at 37° C. in a 5% CO₂ humidified incubator) orembryonated chicken eggs (e.g., in a stationary incubator at 37° C. with55% relative humidity).

The swine influenza viruses used in accordance with the invention may beselected from naturally occurring strains, variants or mutants;mutagenized viruses (e.g., viruses generated by exposure to mutagens,repeated passages and/or passage in non-permissive hosts); reassortants;and/or genetically engineered viruses (e.g., using the “reversegenetics” and helper-free plasmid-based techniques) having the desiredphenotype—i.e., an impaired ability to antagonize the cellular IFNresponse. The naturally occurring strains, variants or mutants,reassortments and/or genetically engineered viruses with the desiredinterferon antagonist phenotype can be selected based on differentialgrowth in cells (e.g., cells of a human (e.g., PerC6, a producer cellline derived from human embryonic retinoblasts transformed with the E1region of Adenovirus 5), mouse, chicken (e.g., chicken embryofibroblasts), rat, birds, or pig (e.g., PK(D1) cells, PK(15) cells, PK13cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells,PT-K75 cells, PK-2a/CL 13 or SJPL cells)) in other assays describedbelow. In certain embodiments, the swine influenza viruses of theinvention are genetically engineered viruses. In other embodiments, theswine influenza viruses of the invention are not naturally occurringstrains, variants or mutants and/or reassortments.

In a specific embodiment, an attenuated swine influenza virus of theinvention comprises a genome comprising a mutation in a swine influenzavirus NS1 gene that diminishes the ability of the NS1 gene product toantagonize a cellular interferon response, and permits the attenuatedvirus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001,0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007,0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 toapproximately 100 fold, approximately 5 to approximately 80 fold,approximately 20 to approximately 80 fold, or approximately 40 toapproximately 80 fold, approximately 1 to approximately 10 fold,approximately 1 to approximately 5 fold, approximately 1 toapproximately 4 fold, approximately 1 to approximately 3 fold,approximately 1 to approximately 2 fold, approximately 3 toapproximately 15 fold, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100fold lower than wild-type swine influenza virus in cells (e.g., cells ofa human (e.g., PerC6, a producer cell line derived from human embryonicretinoblasts transformed with the E1 region of Adenovirus 5), mouse,chicken (e.g., chicken embryo fibroblasts), rat, birds, or pig (e.g.,PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells,LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 or SJPLcells)) as determined by a hemagglutination assay of BALF from pigs orsupernatants of pig cells approximately 2 to 10 days, 3 to 7 days, 3 to5 days, or 2, 3, 5, 6, 7, 8, 9, 10 days post-infection or when theviruses are plagued on Madin-Darby canine kidney (MDCK) cells. In oneembodiment, the growth of an attenuated swine influenza virus of theinvention is compared to a particular standard or reference, e.g.,wild-type swine influenza virus A/Swine/Texas/4199-2/98. In accordancewith these embodiments, the attenuated virus may be geneticallyengineered to contain or express non-swine influenza virus nucleic acidsequences such, e.g., an epitope of a foreign pathogen or a tumorantigen. Preferably, the non-swine influenza virus sequences do notinclude a nucleic acid sequence that alters the attenuated phenotype ofthe virus. Accordingly, nucleic acid sequences encoding proteins,polypeptides or peptides with interferon antagonizing activity arepreferably not engineered into a swine influenza virus.

The invention provides attenuated swine influenza viruses comprising agenome comprising at least two, at least three, at least four or moremutations in two, three, four or more swine influenza virus genes,wherein at least one of the mutations is in the NS1 gene and contributesto or is responsible (directly or indirectly) for the attenuation of thevirus and/or the diminished ability of the virus to antagonize acellular interferon response. In one embodiment, an attenuated swineinfluenza virus of the invention comprises a genome comprising at leasttwo, at least three, at least four or more mutations in two, three, fouror more swine influenza virus genes, wherein at least one of themutations is in the NS1 gene and is responsible for the diminishedability of the NS1 gene product to antagonize a cellular interferonresponse, and permits the attenuated virus, at a MOI of between 0.0005and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers betweenapproximately 1 to approximately 100 fold, approximately 5 toapproximately 80 fold, approximately 20 to approximately 80 fold, orapproximately 40 to approximately 80 fold, approximately 1 toapproximately 10 fold, approximately 1 to approximately 5 fold,approximately 1 to approximately 4 fold, approximately 1 toapproximately 3 fold, approximately 1 to approximately 2 fold,approximately 3 to approximately 15 fold or approximately 1, 2, 3, 4, 5,6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 fold lower than wild-type swine influenza virus in cells(e.g., cells of a human (e.g., PerC6, a producer cell line derived fromhuman embryonic retinoblasts transformed with the E1 region ofAdenovirus 5), mouse, chicken (e.g., chicken embryo fibroblasts), rat,birds, or pig (e.g., PK(D1) cells, PK(15) cells, PK13 cells, NSK cells,LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells,PK-2a/CL 13 or SJPL cells)), as determined by, e.g., hemagglutinationassays, approximately 2 to 10 days, 3 to 7 days, 3 to 5 days, or 2, 3,5, 6, 7, 8, 9, 10 days post-infection when the viruses are propagatedunder the same conditions.

In a specific embodiment, an attenuated swine influenza virus of theinvention comprises a genome comprising at least two, at least three, atleast four or more mutations in two, three, four or more swine influenzavirus genes, wherein at least one of the mutations is in the NS1 geneand is responsible for the diminished ability of the NS1 gene product toantagonize a cellular interferon response, and permits the attenuatedvirus, at a MOI of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05,0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, togrow to titers between approximately 1 to approximately 100 fold,approximately 5 to approximately 80 fold, approximately 20 toapproximately 80 fold, or approximately 40 to approximately 80 fold,approximately 1 to approximately 10 fold, approximately 1 toapproximately 5 fold, approximately 1 to approximately 4 fold,approximately 1 to approximately 3 fold, approximately 1 toapproximately 2 fold, approximately 3 to approximately 15 fold orapproximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold lower than wild-typeswine influenza virus in pig cells, as determined by, e.g.,hemagglutination assays, approximately 2 to 10 days, 3 to 7 days, 3 to 5days, or 2, 3, 5, 6, 7, 8, 9, 10 days post-infection when the virusesare propagated under the same conditions (e.g., in MDCK cells). Inanother embodiment, an attenuated swine influenza virus of the inventioncomprises a genome comprising at least two, three, four or moremutations in two, three, four or more swine influenza virus genes,wherein at least one of the mutations is in the NS1 gene and isresponsible for the attenuation of the virus, and permits the attenuatedvirus, at a MOI of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05,0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, togrow to titers between approximately 1 to approximately 100 fold,approximately 5 to approximately 80 fold, approximately 20 toapproximately 80 fold, or approximately 40 to approximately 80 fold,approximately 1 to approximately 10 fold, approximately 1 toapproximately 5 fold, approximately 1 to approximately 4 fold,approximately 1 to approximately 3 fold, approximately 1 toapproximately 2 fold, approximately 3 to approximately 15 fold orapproximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold lower than wild-typeswine influenza virus in pig cells, as determined by, e.g.,hemagglutination assays, approximately 2 to 10 days, 3 to 7 days, 3 to 5days, or 2, 3, 5, 6, 7, 8, 9, 10 days post-infection when the virusesare propagated under the same conditions (e.g., in MDCK cells). Inaccordance with these embodiments, the attenuated virus has an impairedinterferon antagonist phenotype and may be genetically engineered tocontain or express non-swine influenza virus nucleic acid sequences,such as, e.g., an epitope of a foreign pathogen (e.g., epitopes ofporcine reproductive and respiratory syndrome virus, porcine cytomegalovirus, porcine respiratory corona virus, porcine encephalomyocarditisvirus, porcine epidemic diarrhea and antigenic determinants of non-viralswine pathogens such as bacteria, including, but not limited to,Brucella suis, and parasites, including, but not limited to, roundworms(Ascaris suum), whipworms (Trichuris suis), or a tumor antigen such ascarcinoembryonic antigen (CEA), breast cancer antigen such as EGFR(epidermal growth factor receptor), HER2 antigen (p185^(HER2)), HER2 neuepitope, cancer antigen-50 (CA-50), cancer antigen 15-3 (CA15-3)associated with breast cancer, carcinoma associated antigen (CAA),melanoma antigen, and melanoma associated antigens 100, 25, and 150).Preferably, the non-swine influenza virus sequences (heterologoussequences) do not include a nucleic acid sequence that alters theattenuated phenotype of the virus. Accordingly, nucleic acid sequencesencoding proteins, polypeptides or peptides with interferon antagonizingactivity are preferably not engineered into the swine influenza virus.

Mutations in the swine influenza virus NS1 gene comprise (alternatively,consist of) any mutation that results in the desired phenotype (i.e., animpaired ability to antagonize a cellular interferon response). Examplesof the type of mutations that can be included in or introduced into aswine influenza virus NS1 gene include, but are not limited to,deletions, substitutions, insertions and combinations thereof. One ormore mutations can be located anywhere throughout the NS1 gene, i.e., inthe regulatory, non-coding and/or coding regions (e.g., theamino-terminus and/or the carboxy-terminus or somewhere in between). Ina specific embodiment, an attenuated swine influenza virus of theinvention comprises a genome comprising a swine influenza virus NS1 genewith a mutation (e.g., a deletion or substitution) at the aminoterminus. In a preferred embodiment, an attenuated swine influenza virusof the invention comprises a genome comprising a swine influenza NS1gene with a mutation (e.g., a deletion or substitution, preferably adeletion) at the carboxy terminus. In another embodiment, an attenuatedswine influenza virus of the invention comprises a genome comprising amutation in a swine influenza virus NS1 gene resulting in a deletionconsisting of 5, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 100, 105, 110, 115, 119,120, 121, 125, 130, 135, 140, 145, 146, 147, 148, 150, 155, 160, 165,170 or 175 amino acid residues from the carboxy terminus of NS1, or adeletion of between 5-170, 25-170, 50-170, 100-170, 90-160, 100-160 or105-160, 90-150, 5-75, 5-50 or 5-25 amino acid residues from the carboxyterminus. In another embodiment, an attenuated swine influenza virus ofthe invention comprises a genome comprising a mutation in a swineinfluenza virus NS1 gene resulting in a deletion of all amino acidresidues of the NS1 gene product except amino acid residues 1-126, aminoacid residues 1-120, amino acid residues 1-115, amino acid residues1-110, amino acid residues 1-100, amino acid residues 1-99, amino acidresidues 1-95, amino acid residues 1-85, amino acid residues 1-80, aminoacid residues 1-75, amino acid residues 1-73, amino acid residues 1-70,amino acid residues 1-65 or amino acid residues 1-60, wherein the aminoterminal amino acid is number 1. In accordance with these embodiments,the attenuated swine influenza virus is preferably geneticallyengineered. In a preferred embodiment, an attenuated swine influenzavirus of the invention is TX/98/del 126, TX/98/del 99 or TX/98/del 73.

The attenuated swine influenza virus of the present invention may be achimeric virus that expresses a heterologous sequence. Preferably, theheterologous sequence is not a nucleic acid sequence that alters theattenuated phenotype of the virus. Accordingly, preferably, nucleic acidsequences encoding proteins, polypeptides or peptides with interferonantagonizing activity are not engineered into the swine influenza virus.In certain embodiments, the chimeric virus expresses a tumor antigen. Inother embodiments, the chimeric virus expresses an epitope of a foreignpathogen.

An attenuated swine influenza virus having the desired phenotype canitself be used as the active ingredient in vaccine, pharmaceutical orimmunogenic formulations. Alternatively, the attenuated swine influenzavirus can be used as the vector or “backbone” of recombinantly producedvaccines or immunogenic formulations. To this end, the “reversegenetics” technique or helper-free plasmid approach can be used toengineer mutations or introduce foreign epitopes into the attenuatedswine influenza virus, which would serve as the “parental” strain. Inthis way, vaccines can be designed for immunization against strainvariants, or in the alternative, against completely different infectiousagents or disease antigens (e.g., tumor antigens). For example, theattenuated swine influenza virus can be engineered to expressneutralizing epitopes of other preselected strains. Alternatively,epitopes of other viruses can be built into the attenuated swineinfluenza virus. Alternatively, epitopes of non-viral infectiouspathogens (e.g., parasites, bacteria, fungi) can be engineered into theswine influenza virus.

In a particular embodiment, reassortment techniques can be used totransfer the attenuated phenotype from a parental swine influenza virusstrain (a natural mutant, a mutagenized virus, or a geneticallyengineered virus) to a different virus strain (a wild-type virus, anatural mutant, a mutagenized virus, or a genetically engineered virus).In accordance with this embodiment, the “reverse genetics technique” orhelper-free plasmid approach can be used to engineer mutations orintroduce foreign epitopes into the attenuated swine influenza virus,which would serve as the “parental strain”.

The present invention provides methods for vaccinating subjectscomprising administering a vaccine formulation comprising an attenuatedswine influenza virus comprising a mutation in a swine influenza NS1gene that diminishes the ability of the NS1 gene product to antagonizethe cellular interferon response, and a physiologically effectiveexcipient. In one embodiment, the present invention provides a methodcomprising administering an effective amount of a vaccine formulation ofthe invention. In certain embodiments, the dose of the vaccineformulation administered is between about 10² to about 10⁸, about 10³ toabout 10⁷, or about 10⁴ to about 5×10⁶ pfu. In other embodiments, thedose of the vaccine formulation administered is 10², 5×10², 10³, 5×10³,10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷ or 10⁸ pfu. In specificembodiments, the subject is a donkey, zebra, camel, dog, avian (e.g., aduck). In a preferred embodiment, the subject is a pig.

The present invention provides immunogenic formulations comprising anattenuated swine influenza virus of the invention and methods forinducing an immune response for the treatment, management or preventionof a swine influenza virus infection or a condition or symptomassociated therewith, an infection, other than a swine influenza virusinfection, or a condition or symptom associated therewith, or acondition in which the attenuated swine influenza viruses can be used asa vector to induce an immune response to a particular antigen associatedwith the condition, comprising administering to a subject an immunogenicformulation of the invention. In one embodiment, the present inventionprovides a method for inducing an immune response for the treatment,management or prevention of a swine influenza virus infection or acondition or symptom associated therewith, an infection, other than aswine influenza virus infection, or a condition or symptom associatedtherewith, or a condition in which the attenuated swine influenzaviruses can be used as a vector to induce an immune response to aparticular antigen associated with the condition, comprisingadministering to a subject an effective amount of an immunogenicformulation of the invention. In certain embodiments, the dose of animmunogenic formulation of the invention administered to a subject isbetween about 10² to about 10⁸, about 10³ to about 10⁷, or about 10⁴ toabout 5×10⁶ pfu or about 10⁴ to about 10⁷ pfu. In specific embodiments,the immunogenic formulation administered to a subject has an attenuatedswine influenza virus concentration of about 10⁴ to about 10⁷ pfu/ml. Inother embodiments, the dose of an immunogenic formulation of theinvention administered to a subject is 10², 5×10², 10³, 5×10³, 10⁴,5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷ or 10⁸ pfu. In specificembodiments, the subject is a donkey, zebra, camel, dog, avian (e.g., aduck). In a preferred embodiment, the subject is a pig.

The attenuated swine influenza viruses, which induce robust IFNresponses in subjects, may also be used in pharmaceutical formulationsfor the prophylaxis or treatment of infections and IFN-treatablediseases such as cancer. In this regard, the tropism of the attenuatedswine influenza virus can be altered to target the virus to a desiredtarget organ, tissue or cells in vivo or ex vivo. Using this approach,the IFN response can be induced locally, at the target site, thusavoiding or minimizing the side effects of systemic IFN therapy. To thisend, the attenuated swine influenza virus can be engineered to express aligand specific for a receptor of the target organ, tissue or cells.

The present invention provides methods for preventing, managing ortreating an infection, or IFN-treatable disease in a subject (e.g., apig), other than a swine influenza viral infection, or IFN-treatabledisease caused by swine influenza virus, comprising administering apharmaceutical formulation of the invention. In one embodiment, thepresent invention provides a method for preventing, managing or treatingan infection or IFN-treatable disease in a subject, other than a swineinfluenza viral infection or IFN-treatable disease caused by swineinfluenza virus, comprising administering to a subject an effectiveamount of a pharmaceutical formulation of the invention. In certainembodiments, the dose of the pharmaceutical formulation administered tothe subject is between about 10² to about 10¹², about 10² to about 10¹⁰,about 10² to about 10⁸, about 10³ to about 10⁹, about 10³ to about 10⁷,about 10⁴ to about 10⁸, about 10⁴ to about 5×10⁶, about 10⁴ to about10⁷, or about 10⁴ to about 10¹² pfu. In specific embodiments, thepharmaceutical formulation administered to the subject has an attenuatedinfluenza virus concentration from about 10⁴ to about 10¹² pfu/ml. Inother embodiments, the dose of the pharmaceutical formulationadministered is 10², 5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶,5×10⁶, 10⁷, 5×10⁷, 10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹,5×10¹¹ or 10¹² pfu. In specific embodiments, the subject is a donkey,zebra, camel, dog, avian (e.g., a duck). In a preferred embodiment, thesubject is a pig.

The present invention provides methods for preventing, managing ortreating cancer in a subject comprising administering a pharmaceuticalformulation of the invention. In one embodiment, the present inventionprovides a method for preventing, managing or treating cancer in asubject comprising administering to a subject an effective amount of apharmaceutical formulation of the invention. In certain embodiments, thepharmaceutical formulation comprises a swine influenza virus comprisinga tumor antigen. In certain embodiments, the dose of the pharmaceuticalformulation administered to the subject is between about 10² to about10¹², about 10² to about 10¹⁰, about 10² to about 10⁸, about 10³ toabout 10⁹, about 10³ to about 10⁷, about 10⁴ to about 10⁸, about 10⁴ toabout 5×10⁶ pfu or about 10⁴ to about 10¹² pfu. In other embodiments,the dose of the pharmaceutical formulation administered is 10², 5×10²,10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷, 10⁸, 5×10⁸,1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or 10¹² pfu. In specificembodiments, the subject is a donkey, zebra, camel, dog, avian (e.g., aduck). In a preferred embodiment, the subject is a pig.

Applicants have demonstrated that attenuated swine influenza viruseswith impaired interferon antagonist activity replicate in vivogenerating titers that are lower than detected with wild-type swineinfluenza viruses, but that are sufficient to induce immunological andcytokine responses. Thus, mutations which diminish but do not abolishthe IFN antagonist activity of the swine influenza virus are preferredfor vaccine, immunologic, and pharmaceutical formulations. Such virusescan be selected for growth in both conventional and non-conventionalsubstrates, and for intermediate virulence.

The present invention provides cells containing (alternatively,comprising) swine influenza viruses of the present invention. In aspecific embodiment, a cell contains/comprises a swine influenza virusthat is genetically engineered. In certain embodiments, the attenuatedswine influenza virus contained in the cell is engineered to encode anepitope derived from another pathogen (e.g., another virus) or a tumorantigen. In accordance with this embodiment, the genome of theattenuated swine influenza virus may comprise at least one segmentderived from a different virus. Any cell can be infected with, containor comprise an attenuated swine influenza virus of the inventionincluding, but not limited to, MDCK cells, PK cells, Vero cells, primaryhuman umbilical vein endothelial cells (HUVEC), H292 human epithelialcell line, HeLa cells, and swine embryonic kidney cells RES. In apreferred embodiment, the cell is a pig cell or a pig cell line. Incertain embodiments, the cell (e.g., a pig cell or pig cell line) isinterferon deficient.

In certain other embodiments, the swine influenza viruses of theinvention are contained in 10 day-old, 6 to 9 day-old, 6 to 8 day-old,or 6 to 7 day-old embryonated chicken eggs. In a specific embodiment,the viruses are contained in allantoic cavity of such eggs. In anotherspecific embodiment, the viruses are contained in the amniotic cavity ofsuch eggs.

The invention encompasses the use of substrates such as cells, celllines and embryonated eggs, to propagate the attenuated swine influenzaviruses of the invention. In one embodiment, the invention providesmethods for vaccine production and pharmaceutical production comprisingpropagating in a substrate an attenuated swine influenza virus of thepresent invention and collecting progeny virus, wherein the substrate isa cell, cell line or embryonated egg. In certain embodiments, thesubstrate is an IFN deficient system. Exemplary IFN-deficient systemsinclude, but are not limited to, young embryonated eggs (e.g., 6 to 10days old, 6 to 9 days old, 6 to 8 days old or 6 to 7 days oldembryonated eggs), and IFN-deficient cell lines (such as VERO cells orgenetically engineered cell lines such as STAT1 knockouts). Embryonatedeggs or cell lines pretreated with compounds that inhibit the IFN system(including drugs, antibodies, antisense, ribozymes, etc.) can also beused as an IFN-deficient system. Further, eggs deficient in the IFNsystem, e.g., eggs produced by STAT1 negative birds, especially fowl,including but not limited to transgenic chickens, ducks or turkeys, maybe used as an IFN-deficient system.

3.1 Terminology

As used herein, the term “about” or “approximately” when used inconjunction with a number refers to any number within 1, 5 or 10% of thereferenced number.

As used herein, the phrase “amino-terminus” of NS1 refer to the aminoacids from the amino terminal amino acid residue (amino acid residue 1)through amino acid residue 115, amino acid residues 1 through 100, aminoacid residues 1 through 75, amino acid residues 1 through 50, amino acidresidues 1 through 25, or amino acid residues 1 through 10 of the swineinfluenza viral NS1 protein.

As used herein, the phrase “carboxy-terminus” of NS1 refer to amino acidresidues 116 through the carboxy terminal amino acid residue, amino acidresidues 101 through the carboxy terminal amino acid residue, amino acidresidues 76 through the carboxy terminal amino acid residue, amino acidresidues 51 through the carboxy terminal amino acid residue, or aminoacid residues 26 through the carboxy terminal amino acid residue of theswine influenza viral NS1 protein, when the amino-terminus of NS1 isamino acid residues 1 through amino acid residue 115, amino acidresidues 1 through 100, amino acid residues 1 through 75, amino acidresidues 1 through 50, or amino acid residues 1 through 25,respectively, of the swine influenza viral NS1 protein. Deletions fromthe carboxy terminus can include deletions consisting of 5, preferably10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75, 80, 85, 90,95, 99, 100, 105, 110, 115, 120, 125, 126, 130, 135, 140, 145, 150, 155,160, 165, 170 or 175 amino acid residues from the carboxy terminus ofNS1.

As used herein, the term “cytokine receptor modulator” refers to anagent which modulates the phosphorylation of a cytokine receptor, theactivation of a signal transduction pathway associated with a cytokinereceptor, and/or the expression of a particular protein such as acytokine Such an agent may directly or indirectly modulate thephosphorylation of a cytokine receptor, the activation of a signaltransduction pathway associated with a cytokine receptor, and/or theexpression of a particular protein such as a cytokine Thus, examples ofcytokine receptor modulators include, but are not limited to, cytokines,fragments of cytokines, fusion proteins, and antibodies thatimmunospecifically binds to a cytokine receptor or a fragment thereof.Further, examples of cytokine receptor modulators include, but are notlimited to, peptides, polypeptides (e.g., soluble cytokine receptors),fusion proteins and antibodies that immunospecifically binds to acytokine or a fragment thereof.

As used herein, the term “effective amount” refers to the amount of atherapy (e.g., a prophylactic or therapeutic agent) which is sufficientto reduce and/or ameliorate the severity and/or duration of a condition(e.g., a swine influenza virus infection or a condition or symptomassociated therewith, another infection (e.g., another viral infection),an IFN treatable disease or a condition in which the attenuated swineinfluenza viruses can be used as a vector to induce an immune responseto a particular antigen associated with the condition), prevent theadvancement of a condition (e.g., a swine influenza virus infection or acondition or symptom associated therewith, another infection (e.g.,another viral infection), an IFN treatable disease or a condition inwhich the attenuated swine influenza viruses can be used as a vector toinduce an immune response to a particular antigen associated with thecondition), cause regression of a condition (e.g., a swine influenzavirus infection or a condition or symptom associated therewith, anotherinfection (e.g., another viral infection) or an IFN treatable disease),prevent the recurrence, development, or onset of one or more symptomsassociated with a condition (e.g., a swine influenza virus infection ora condition or symptom associated therewith, another infection (e.g.,another viral infection) or an IFN treatable disease), reduce the titerof swine influenza virus or enhance or improve the prophylactic ortherapeutic effect(s) of another therapy (e.g., prophylactic ortherapeutic agent).

As used herein, the term “epitopes” refers to sites or fragments of apolypeptide or protein having antigenic or immunogenic activity in ananimal, preferably in a mammal, and most preferably in a pig. An epitopehaving immunogenic activity is a site or fragment of a polypeptide orprotein that elicits an antibody response in an animal. An epitopehaving antigenic activity is a site or fragment of a polypeptide orprotein to which an antibody immunospecifically binds as determined byany method well-known to one of skill in the art, for example byimmunoassays.

As used herein, the term “fragment” in the context of a proteinaceousagent refers to a peptide or polypeptide comprising an amino acidsequence of at least 2 contiguous amino acid residues, at least 5contiguous amino acid residues, at least 10 contiguous amino acidresidues, at least 15 contiguous amino acid residues, at least 20contiguous amino acid residues, at least 25 contiguous amino acidresidues, at least 40 contiguous amino acid residues, at least 50contiguous amino acid residues, at least 60 contiguous amino residues,at least 70 contiguous amino acid residues, at least 80 contiguous aminoacid residues, at least 90 contiguous amino acid residues, at least 100contiguous amino acid residues, at least 125 contiguous amino acidresidues, at least 150 contiguous amino acid residues, at least 175contiguous amino acid residues, at least 200 contiguous amino acidresidues, or at least 250 contiguous amino acid residues of the aminoacid sequence of a peptide, polypeptide or protein. In one embodiment, afragment of a full-length protein retains activity of the full-lengthprotein, e.g., IFN antagonist activity. In another embodiment, thefragment of the full-length protein does not retain the activity of thefull-length protein, e.g., IFN antagonist activity.

As used herein, the term “fragment” in the context of a nucleic acidrefers to a nucleic acid comprising an nucleic acid sequence of at least2 contiguous nucleotides, at least 5 contiguous nucleotides, at least 10contiguous nucleotides, at least 15 contiguous nucleotides, at least 20contiguous nucleotides, at least 25 contiguous nucleotides, at least 30contiguous nucleotides, at least 35 contiguous nucleotides, at least 40contiguous nucleotides, at least 50 contiguous nucleotides, at least 60contiguous nucleotides, at least 70 contiguous nucleotides, at leastcontiguous 80 nucleotides, at least 90 contiguous nucleotides, at least100 contiguous nucleotides, at least 125 contiguous nucleotides, atleast 150 contiguous nucleotides, at least 175 contiguous nucleotides,at least 200 contiguous nucleotides, at least 250 contiguousnucleotides, at least 300 contiguous nucleotides, at least 350contiguous nucleotides, or at least 380 contiguous nucleotides of thenucleic acid sequence encoding a peptide, polypeptide or protein. In apreferred embodiment, a fragment of a nucleic acid encodes a peptide orpolypeptide that retains activity of the full-length protein, e.g., IFNantagonist activity. In another embodiment, the fragment of thefull-length protein does not retain the activity of the full-lengthprotein, e.g., IFN antagonist activity.

As used herein, the phrase “heterologous sequence” refers to anysequence nucleic acid or protein, polypeptide or peptide sequence whichis not normally found or in nature associated in nature with a nucleicacid or protein, polypeptide or peptide sequence of interest. Forexample, a “heterologous sequence” may refer to a sequence derived froma different species.

As used herein, the term “in combination” refers to the use of more thanone therapy (e.g., more than one prophylactic agent and/or therapeuticagent). The use of the term “in combination” does not restrict the orderin which therapies (e.g., prophylactic and/or therapeutic agents) areadministered to a subject with a condition (e.g., a swine influenzavirus infection or a condition or symptom associated therewith, anotherinfection (e.g., another viral infection) or an IFN treatable disease).A first therapy (e.g., a first prophylactic or therapeutic agent) can beadministered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of a second therapy (e.g., a secondprophylactic or therapeutic agent) to a subject with a condition (e.g.,a swine influenza virus infection or a condition or symptom associatedtherewith, another infection (e.g., another viral infection) or an IFNtreatable disease).

As used herein, the phrase “interferon antagonist activity” refers to aprotein or polypeptide, or fragment, derivative, or analog thereof thatreduces or inhibits the cellular interferon immune response. Inparticular, a protein or polypeptide, or fragment, derivative, or analogthereof (e.g., swine influenza virus NS1) that has interferon antagonistactivity reduces or inhibits interferon expression and/or activity. In aspecific embodiment, the phrase “interferon antagonist activity” refersto a swine influenza virus protein or polypeptide, or fragment,derivative, or analog thereof that reduces or inhibits the cellularinterferon immune response. A swine influenza viral protein orpolypeptide with interferon antagonist activity may preferentiallyaffect the expression and/or activity of one or two types of interferon(IFN). In one embodiment, the expression and/or activity of IFN-α isaffected. In another embodiment, the expression and/or activity of IFN-βis affected. In one embodiment, the expression and/or activity of IFN-γis affected. In certain embodiments, the expression and/or activity ofIFN-β and/or IFN-γ is reduced 5-10%, 10-20%, 20-30%, 30-40%, 40-50%,50-60%, 60-70%, 70-80%, 80-90% or more by protein, polypeptide, etc.with an interferon antagonist activity when compared to a control (e.g.,PBS or a protein without interferon antagonist activity) inIFN-competent systems, e.g., a wild-type cell or animal under the sameconditions. In certain embodiments, the expression and/or activity ofIFN-β and/or IFN-γ is reduced approximately 1 to approximately 100 fold,approximately 5 to approximately 80 fold, approximately 20 toapproximately 80 fold, approximately 1 to approximately 10 fold, orapproximately 1 to approximately 5 fold, or approximately 40 toapproximately 80 fold, or 1, 2, 3, 4, 5, 7, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold by protein,polypeptide, etc. with an interferon antagonist activity when comparedto a control (e.g., PBS or a protein without interferon antagonistactivity) in IFN-competent systems under the same conditions.

As used herein, the phrases “IFN-deficient systems” or “IFN-deficientsubstrates” refer to systems, e.g., cells, cell lines and animals, suchas pigs, mice, chickens, turkeys, rabbits, rats, etc., which do notproduce IFN or produce low levels of IFN (i.e., a reduction in IFNexpression of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%,70-80%, 80-90% or more when compared to IFN-competent systems under thesame conditions), do not respond or respond less efficiently to IFN,and/or are deficient in the activity of one or more antiviral genesinduced by IFN.

As used herein, the phrase “IFN-inducing phenotype” refers to aphenotype whereby a virus demonstrates an increased cellular interferonresponse compared to a wild-type virus, which typically inhibits orreduces cellular interferon mediated responses.

As used herein, the phrases “IFN treatable disorders”, “IFN treatablediseases” and analogous phrases refer to conditions that arepreventable, treatable, managed or ameliorated by the administration ofIFN, either IFN-α, β, γ or any combination thereof. The IFN treatabledisorders need not be limited to swine, if swine influenza virus infectsother species. Examples of IFN treatable disorders in pigs include, butare not limited to, foot and mouth disease, porcine Haemophiluspneumonia (PHP, and other disorders caused by infection with, e.g.,infection by A. pleuropneumoniae, P. haemolytica, P. multocida, H.somnus and A. suis.

As used herein, the phrase “intermediate” phenotype with respect toIFN-antagonist activity refers to a phenotype which can stimulate arobust immune response, while being attenuated because viruses with anintermediate phenotype cannot overcome the host IFN response. Inparticular, an intermediate phenotype can stimulate an immune responseand inhibit/reduce IFN only to the extent that it allows fewer rounds ofvirus replication or lower numbers of virus particles being producedcompared to wild-type virus, as determined by techniques well-known inthe art (e.g., as measured by lower plaque titer or hemagglutinationassays).

As used herein, the term “isolated”, in the context of viruses, refersto a virus that is derived from a single parental virus. A virus can beisolated using routine methods known to one of skill in the artincluding, but not limited to, those based on plaque purification andlimiting dilution.

As used herein, the terms “manage,” “managing,” and “management” referto the beneficial effects that a subject derives from a therapy (e.g., aprophylactic or therapeutic agent), which does not result in a cure ofthe disease. In certain embodiments, a subject is administered one ormore therapies (e.g., one or more prophylactic or therapeutic agents) to“manage” a disease so as to prevent the progression or worsening of thedisease.

As used herein, the phrase “multiplicity of infection” or “MOI” is theaverage number of virus per infected cell. The MOI is determined bydividing the number of virus added (ml added×PFU) by the number of cellsadded (ml added×cells/ml).

As used herein, the phrase “non-swine influenza virus” in the context ofswine influenza viruses containing or comprising non-swine influenzavirus sequences refers to any influenza virus strain or isolatecomprising a sequence (e.g., a nucleic acid or amino acid sequence)heterologous to swine influenza virus, i.e, the sequence is not found tonaturally in association with swine influenza virus.

As used herein, the phrase “NS1 gene” refers to the gene which encodesthe nonstructural protein (NS1) in influenza. NS1 is one of the eightmolecules encoded by the segmented genome of influenza A and otherviruses. A “swine influenza virus NS1 gene” is an NS1 gene isolated froma swine influenza virus. Representative swine NS1 genes can be found inpublic sequence databases such as Genbank and include, but are notlimited to, Genbank Accession No. AJ293939(A/swine/Italy/13962/95(H3N2)) and Genbank Accession No. AJ344041(A/swine/Cotes d'Armor/1121/00(H1N1)). A “NS1 gene product” refers to agene product (e.g., a RNA or protein) encoded by a NS1 gene. In the caseof a protein, the NS1 gene product is full-length and has wild-type NS1activity, (e.g., from Influenza A/swine/Texas/4199-2/98. A “swineinfluenza virus NS1 gene product” refers to a gene product (e.g., a RNAor protein) encoded by a swine influenza virus NS1 gene. In the case ofa protein, the swine influenza virus NS1 gene product is full-length andhas wild-type swine influenza virus NS1 activity, e.g., from InfluenzaA/swine/Texas/4199-2/98.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the inhibition of the development or onset of a condition (e.g, aswine influenza virus infection or a condition associated therewith, aninfection other than a swine influenza virus infection or a conditionassociated therewith, an IFN-treatable disease or a condition in whichthe attenuated swine influenza viruses can be used as a vector to inducean immune response to a particular antigen associated with thecondition), or the prevention of the recurrence, onset, or developmentof one or more symptoms of a condition (e.g., a swine influenza virusinfection or a condition associated therewith, an infection other than aswine influenza virus infection or a condition associated therewith, oran IFN-treatable disease), in a subject resulting from theadministration of a therapy (e.g., a prophylactic or therapeutic agent),or the administration of a combination of therapies (e.g., a combinationof prophylactic or therapeutic agents).

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) which can be used in the prevention of a conditionor a symptom thereof (e.g., a swine influenza virus infection or acondition or symptom associated therewith, an infection other than aswine influenza virus infection or a condition or symptom associatedtherewith, an IFN treatable disease or a condition in which theattenuated swine influenza viruses can be used as a vector to induce animmune response to a particular antigen associated with the condition).Preferably, a prophylactic agent is an agent which is known to be usefulto or has been or is currently being used to the prevent or impede theonset, development, progression and/or severity of a swine influenzavirus infection or a condition or symptom associated therewith, aninfection other than a swine influenza virus infection or a condition orsymptom associated therewith, an IFN treatable disease or a condition inwhich the attenuated swine influenza viruses can be used as a vector toinduce an immune response to a particular antigen associated with thecondition.

As used herein, the term “prophylactically effective amount” refers tothe amount of a therapy (e.g., prophylactic agent) which is sufficientto result in the prevention of the development, recurrence, or onset ofa condition or a symptom thereof (e.g., a swine influenza virusinfection or a condition or symptom associated therewith, an infectionother than a swine influenza virus infection or a condition or symptomassociated therewith, an IFN treatable disease or a condition in whichthe attenuated swine influenza viruses can be used as a vector to inducean immune response to a particular antigen associated with thecondition) or to enhance or improve the prophylactic effect(s) ofanother therapy (e.g., a prophylactic agent).

As used herein, the phrase “purified” in the context of viruses refersto a virus which is substantially free of cellular material and culturemedia from the cell or tissue source from which the virus is derived.The language “substantially free of cellular material” includespreparations of virus in which the virus is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. Thus, virus that is substantially free of cellular materialincludes preparations of protein having less than about 30%, 20%, 10%,or 5% (by dry weight) of cellular protein (also referred to herein as a“contaminating protein”). The virus is also substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the virus preparation. A virus can bepurified using routine methods known to one of skill in the artincluding, but not limited to, chromatography and centrifugation.

As used herein, the terms “subject” or “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refers to an animal (e.g., birds, reptiles, and mammals), preferably amammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig,horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey,chimpanzee, and a human). In certain embodiments, the subject or patienthas a swine influenza virus infection. In certain embodiments, themammal is 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to10 years old, 10 to 15 years old, 15 to 20 years old, 20 to 25 yearsold, 25 to 30 years old, 30 to 35 years old, 35 to 40 years old, 40 to45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60 yearsold, 60 to 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years oldor 95 to 100. In a preferred embodiment, the subject or patient is apig. In certain embodiments, the pig is 0 to 6 months old, 6 to 12months old, 1 to 5 years old, 5 to 10 years old or 10 to 15 years old.The natural lifespan of a pig is 10-15 years.

As used herein, “swine influenza virus” refers to a type A or type Cinfluenza virus from the family orthomyxovirus that causes swineinfluenza. While orthomyxovirus has three groups: type A, type B andtype C, only type A and type C influenza viruses infect pigs. Subtypesof swine influenza virus include H1N1, H1N2, H3N2, and H3N1. H9N2 andH5N1 can also be found in pigs. In certain embodiments, a swineinfluenza virus is an influenza virus that has been isolated from swine.In a preferred embodiment, a swine influenza virus contains a swine NS1gene. Representative swine NS1 genes can be found in public sequencedatabases such as Genbank and include, but are not limited to, GenbankAccession No. AJ293939 (A/swine/Italy/13962/95(H3N2)) and GenbankAccession No. AJ344041 (A/swine/Cotes d'Armor/1121/00(H1N1)). Examplesof swine influenza virus variants include, but are not limited to,A/Swine/Colorado/1/77, A/Swine/Colorado/23619/99, A/Swine/Coted'Armor/3633/84, A/Swine/Cote d'Armor/3633/84,A/Swine/England/195852/92, A/Swine/Finistere/2899/82, A/Swine/HongKong/10/98, A/Swine/Hong Kong/9/98, A/Swine/Hong Kong/81/78,A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01,A/Swine/Illinois/21587/99, A/Swine/Indiana/1726/88,A/Swine/Indiana/9K035/99, A/Swine/Indiana/P12439/00, A/Swine/Iowa/30,A/Swine/Iowa/15/30, A/Swine/Iowa/533/99, A/Swine/Iowa/569/99,A/Swine/Iowa/3421/90, A/Swine/Iowa/8548-1/98, A/Swine/Iowa/930/01,A/Swine/Iowa/17672/88, A/Swine/Italy/1513-1/98, A/Swine/Italy/1523/98,A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00,A/Swine/Minnesota/593/99, A/Swine/Minnesota/9088-2/98,A/Swine/Nebraska/1/92, A/Swine/Nebraska/209/98,A/Swine/Netherlands/12/85, A/Swine/North Carolina/16497/99,A/Swine/North Carolina/35922/98, A/Swine/North Carolina/93523/01,A/Swine/North Carolina/98225/01, A/Swine/Oedenrode/7C/96,A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/99,A/Swine/Oklahoma/18089/99, A/Swine/Ontario/01911-1/99,A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97,A/Swine/Ontario/97, A/Swine/Quebec/192/81, A/Swine/Quebec/192/91,A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Tennessee/24/77,A/Swine/Texas/4199-2/98, A/Swine/Wisconsin/125/97,A/Swine/Wisconsin/136/97, A/Swine/Wisconsin/163/97,A/Swine/Wisconsin/164/97, A/Swine/Wisconsin/166/97,A/Swine/Wisconsin/168/97, A/Swine/Wisconsin/235/97,A/Swine/Wisconsin/238/97, A/Swine/Wisconsin/457/98,A/Swine/Wisconsin/458/98, A/Swine/Wisconsin/464/98 andA/Swine/Wisconsin/14094/99.

As used herein, the term “synergistic” refers to a combination oftherapies (e.g., prophylactic or therapeutic agents) which is moreeffective than the additive effects of any two or more single therapies(e.g., one or more prophylactic or therapeutic agents). A synergisticeffect of a combination of therapies (e.g., a combination ofprophylactic or therapeutic agents) permits the use of lower dosages ofone or more of therapies (e.g., one or more prophylactic or therapeuticagents) and/or less frequent administration of said therapies to asubject with a condition (e.g., a swine influenza virus infection or acondition or symptom associated therewith, an infection other than aswine influenza virus infection or a condition or symptom associatedtherewith, an IFN treatable disease or a condition in which theattenuated swine influenza viruses can be used as a vector to induce animmune response to a particular antigen associated with the condition).The ability to utilize lower dosages of therapies (e.g., prophylactic ortherapeutic agents) and/or to administer said therapies less frequentlyreduces the toxicity associated with the administration of saidtherapies to a subject without reducing the efficacy of said therapiesin the prevention or treatment of a condition (e.g., a swine influenzavirus infection or a condition or symptom associated therewith, aninfection other than a swine influenza virus infection or a condition orsymptom associated therewith, an IFN treatable disease or a condition inwhich the attenuated swine influenza viruses can be used as a vector toinduce an immune response to a particular antigen associated with thecondition). In addition, a synergistic effect can result in improvedefficacy of therapies (e.g., prophylactic or therapeutic agents) in theprevention or treatment of a condition (e.g., a swine influenza virusinfection or a condition or symptoms associated therewith, an infectionother than a swine influenza virus infection or a condition or symptomassociated therewith, an IFN treatable disease or a condition in whichthe attenuated swine influenza viruses can be used as a vector to inducean immune response to a particular antigen associated with thecondition). Finally, synergistic effect of a combination of therapies(e.g., prophylactic or therapeutic agents) may avoid or reduce adverseor unwanted side effects associated with the use of any single therapy.

As used herein, the term “T cell receptor modulator” refers to an agentwhich modulates the phosphorylation of a T cell receptor, the activationof a signal transduction pathway associated with a T cell receptorand/or the expression of a particular protein such as a cytokine Such anagent may directly or indirectly modulate the phosphorylation of a Tcell receptor, the activation of a signal transduction pathwayassociated with a T cell receptor, and/or the expression of a particularprotein such as a cytokine Examples of T cell receptor modulatorsinclude, but are not limited to, peptides, polypeptides, proteins,fusion proteins and antibodies which immunospecifically bind to a T cellreceptor or a fragment thereof. Further, examples of T cell receptormodulators include, but are not limited to, proteins, peptides,polypeptides (e.g., soluble T cell receptors), fusion proteins andantibodies that immunospecifically bind to a ligand for a T cellreceptor or a fragment thereof.

As used herein, the term “therapeutically effective amount” refers tothe amount of a therapy, which is sufficient to reduce the severity of acondition (e.g., a swine influenza virus infection or a conditionassociated therewith (e.g., influenza induced abortions, depression,inappetance, anorexia, dyspnea, myalgia, or enlarged submandibular lymphnodes), an infection other than a swine influenza virus infection or acondition or symptom associated therewith, an IFN treatable disease or acondition in which the attenuated swine influenza viruses can be used asa vector to induce an immune response to a particular antigen associatedwith the condition), reduce the duration of a condition (e.g., swineinfluenza virus infection or a condition associated therewith, aninfection other than a swine influenza virus infection or a condition orsymptom associated therewith, an IFN treatable disease or a condition inwhich the attenuated swine influenza viruses can be used as a vector toinduce an immune response to a particular antigen associated with thecondition), reduce the titer of swine influenza virus or other virus,reduce the number of other pathogens, ameliorate one or more symptoms ofa condition (e.g., a swine influenza virus infection or a conditionassociated therewith, an infection other than a swine influenza virusinfection or a condition or symptom associated therewith, an IFNtreatable disease or a condition in which the attenuated swine influenzaviruses can be used as a vector to induce an immune response to aparticular antigen associated with the condition), prevent theadvancement of a condition (e.g., a swine influenza virus infection or acondition associated therewith, an infection other than a swineinfluenza virus infection or a condition or symptom associatedtherewith, an IFN treatable disease or a condition in which theattenuated swine influenza viruses can be used as a vector to induce animmune response to a particular antigen associated with the condition),cause regression of a condition (e.g., a swine influenza virus infectionor a condition associated therewith, an infection other than a swineinfluenza virus infection or a condition or symptom associatedtherewith, an IFN treatable disease or a condition in which theattenuated swine influenza viruses can be used as a vector to induce animmune response to a particular antigen associated with the condition),or enhance or improve the therapeutic effect(s) of another therapy.

As used herein, the terms “therapies” and “therapy” can refer to anyprotocol(s), method(s), compositions, formulations, and/or agent(s) thatcan be used in the prevention, treatment, management, or amelioration ofa condition (e.g., a swine influenza virus infection or a condition orsymptom associated therewith, an infection other than a swine influenzavirus infection or a condition or symptom associated therewith, an IFNtreatable disease or a condition in which the attenuated swine influenzaviruses can be used as a vector to induce an immune response to aparticular antigen associated with the condition). In certainembodiments, the terms “therapies” and “therapy” refer to biologicaltherapy, supportive therapy, and/or other therapies useful in treatment,management, prevention, or amelioration of a swine influenza virusinfection or a condition or symptom associated therewith, an infectionother than a swine influenza virus infection or a condition or symptomassociated therewith, an IFN treatable disease or a condition in whichthe attenuated swine influenza viruses can be used as a vector to inducean immune response to a particular antigen associated with thecondition, known to one of skill in the art.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the prevention, treatment,management, or amelioration of a condition or a symptom thereof (e.g., aswine influenza infection or a condition or symptoms associatedtherewith, an infection other than a swine influenza virus infection ora condition or symptom associated therewith, an IFN treatable disease ora condition in which the attenuated swine influenza viruses can be usedas a vector to induce an immune response to a particular antigenassociated with the condition). Preferably, a therapeutic agent is anagent which is known to be useful for, or has been or is currently beingused for the prevention, treatment, management, or amelioration of aswine influenza virus infection or a condition or symptoms associatedtherewith, an infection other than a swine influenza virus infection ora condition or symptom associated therewith, an IFN treatable disease ora condition in which the attenuated swine influenza viruses can be usedas a vector to induce an immune response to a particular antigenassociated with the condition.

As used herein, the terms “treat,” “treatment,” and “treating” refer tothe eradication or control of swine influenza virus replication or thereplication of a pathogen (e.g., a virus) other than swine influenzavirus, the reduction in the titer of swine influenza virus or virusother than swine influenza virus, the reduction in the numbers of apathogen, the reduction or amelioration of the progression, severity,and/or duration of a condition (e.g., a swine influenza virus infectionor a condition associated therewith, an infection other than a swineinfluenza virus infection or a condition or symptom associatedtherewith, an IFN treatable disease or a condition in which theattenuated swine influenza viruses can be used as a vector to induce animmune response to a particular antigen associated with the condition),or the amelioration of one or more symptoms resulting from theadministration of one or more therapies (including, but not limited to,the administration of one or more prophylactic or therapeutic agents).

The term “tumor antigen” as used herein refers to a molecule on a tumorcell that can be specifically recognized by immune T cells orantibodies. A tumor antigen includes those present only on tumor cells(tumor specific antigens) as well as those present on normal cells butexpressed preferentially or aberrantly on tumor cells (tumor associatedantigens). Examples of tumor antigens include, but are not limited to,antigens of sarcoids, prostate cancer, fibrosarcoma,self-differentiation antigens such as oncofetal, or differentiation,antigens which are expressed by malignant cells, including but notlimited to oncofetal antigens such as carcinoembryonio antigens (CEA) ofthe colon, alpha-fetoprotein, the human antigenic counterpart orfunctional equivalent of the 175 kDa murine antigen of transitional cellbladder carcinomas, the melanoma associated antigen p97 or GD3, anddifferentiation antigens of human lung carcinomas such as L6 and L20.

As used herein, the phrase “wild-type swine influenza virus” refers tothe types of an swine virus that are prevalent, circulating naturallyand producing typical outbreaks of disease. Examples of wild-type swineinfluenza viruses include, but are not limited to,A/Swine/Colorado/1/77, A/Swine/Colorado/23619/99, A/Swine/Coted'Armor/3633/84, A/Swine/Cote d'Armor/3633/84,A/Swine/England/195852/92, A/Swine/Finistere/2899/82, A/Swine/HongKong/10/98, A/Swine/Hong Kong/9/98, A/Swine/Hong Kong/81/78,A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01,A/Swine/Illinois/21587/99, A/Swine/Indiana/1726/88,A/Swine/Indiana/9K035/99, A/Swine/Indiana/P12439/00, A/Swine/Iowa/30,A/Swine/Iowa/15/30, A/Swine/Iowa/533/99, A/Swine/Iowa/569/99,A/Swine/Iowa/3421/90, A/Swine/Iowa/8548-1/98, A/Swine/Iowa/930/01,A/Swine/Iowa/17672/88, A/Swine/Italy/1513-1/98, A/Swine/Italy/1523/98,A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00,A/Swine/Minnesota/593/99, A/Swine/Minnesota/9088-2/98,A/Swine/Nebraska/1/92, A/Swine/Nebraska/209/98,A/Swine/Netherlands/12/85, A/Swine/North Carolina/16497/99,A/Swine/North Carolina/35922/98, A/Swine/North Carolina/93523/01,A/Swine/North Carolina/98225/01, A/Swine/Oedenrode/7C/96,A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/99,A/Swine/Oklahoma/18089/99, A/Swine/Ontario/01911-1/99,A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97,A/Swine/Ontario/97, A/Swine/Quebec/192/81, A/Swine/Quebec/192/91,A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Tennessee/24/77,A/Swine/Texas/4199-2/98, A/Swine/Wisconsin/125/97,A/Swine/Wisconsin/136/97, A/Swine/Wisconsin/163/97,A/Swine/Wisconsin/164/97, A/Swine/Wisconsin/166/97,A/Swine/Wisconsin/168/97, A/Swine/Wisconsin/235/97,A/Swine/Wisconsin/238/97, A/Swine/Wisconsin/457/98,A/Swine/Wisconsin/458/98, A/Swine/Wisconsin/464/98 andA/Swine/Wisconsin/14094/99.

4. DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Generation of plasmid-derived Sw/Tx/98 influenza viruseswith NS1 mutated proteins. FIG. 1A depicts a schematic diagram of thewild type and mutated NS influenza gene segments. The NS gene segmentwas modified to create mutated NS1 genes encoding 73, 99 and 126 aa,respectively. NS1 mutations did not affect the sequence of the NEPprotein. Underlined sequences were introduced to generate stop codons(in bold) (SEQ ID NOs: 19, 20 and 21, respectively). FIG. 1B depictsRT-PCR analysis of the rWT and NS1 deletion mutant viruses. Influenzaviral RNA segments were amplified using primers specific for thenon-coding regions of all 8 influenza viral segments. Arrows indicatethe decreasing size of the NS segment. Dots indicate a productcorresponding to the 3′ end of the HA gene segment due to internalbinding of the forward primer.

FIGS. 2A-2D. Characterization of plasmid-derived wild-type and NS1mutant Sw/TX/98 viruses in tissue culture. FIG. 2A shows multi-cyclegrowth curves of wild-type and NS1 deletion mutants in PK-15 cells. FIG.2B shows single cycle growth curves of wild-type and NS1 deletionmutants in PK-15 cells. FIG. 2C shows plaque size in MDCK cells at 3days post infection. FIG. 2D shows a time course of viral proteinexpression. PK-15 cells infected with rWT and NS1 mutant viruses (MOI=2)and labeled with [³⁵S]Met-Cys at the indicated times p.i. Cell extractswere subjected to SDS-PAGE and analyzed by autoradiography.

FIGS. 3A-3C. Induction of type I IFN in PK-15 cells infected withplasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses. FIG. 3A showsa schematic representation of the IFN-bioassay levels of IFN secreted byvirus infected cells in a fluorescent assay. FIG. 3B shows a time courseof type I IFN synthesis. Fresh PK-15 cells were treated for 24 hourswith UV-inactivated supernatants (harvested at different times p.i.)from PK-15 cells which were infected with the indicated viruses,followed by VSV-GFP infection. Sixteen hours post infection, cellsexpressing GFP were visualized by fluorescence microscopy. the inductionof IFN and TNF-α. FIG. 3C shows a RT-PCR analysis of TNF-α, IFN-β- andβ-actin-specific mRNAs in virus-infected PK-15 cells.

FIGS. 4A-4C. Infection of 4-week-old pigs with plasmid-derived wild-typeand NS1 mutant Sw/TX/98 viruses. FIG. 4A shows a bar chart depicting thehistopathological scores of lungs from pigs infected withplasmid-derived TX/98 deletion viruses. Mean percentage (±SEM) of lungsurface with macroscopic lesions on day 5 p.i. FIG. 4B shows microscopiclesions in the medium bronchioles on day 5 p.i. FIG. 4C shows virustiters in bronchoalveolar lavage fluid on day 5 p.i.

FIGS. 5A-5D. Histopathological examination of lungs from pigs infectedwith plasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses,magnification 200×. FIG. 5A shows medium-sized bronchiole from the lungof a non-infected control pig. Epithelial lining is intact, andsurrounding alveoli are normal. FIG. 5B shows severe acute necrotizingbronchiolitis and interstitial pneumonia characteristic of thewidespread lesion induced by infection with rWT TX/98 virus. FIG. 5Cshows one normal bronchiole and one affected bronchiole in earlyrecovery from infection with deletion mutant 1-99 virus, representativeof the less extensive injury induced by infection with this virus. FIG.5D shows normal bronchiole and surrounding alveoli from the lung of apig inoculated with 1-126 virus. No lesions were induced by this virus.

FIGS. 6A-D. Histopathological examination of lungs from pigs infectedwith plasmid-derived wild-type and NS1 mutant Sw/TX/98 viruses,magnification 200×. FIG. 6A shows the epithelial lining of a largerbronchiole from the lung of a non-infected control pig. Epithelial cellsare tall columnar with pseudo-stratified basal nuclei and prominentsurface cilia. FIG. 6B shows the epithelial lining from a largerbronchiole from the lung of a pig infected with TX/98 rWT virus.Epithelial lining is in total disarray due to necrosis and regenerativeproliferation of the epithelial cells. FIG. 6C shows a large bronchioleand smaller branching airway from the lung of a pig infected withdeletion mutant 1-99 virus, representative of the less extensive injuryinduced by this virus. FIG. 6D shows a normal epithelial lining from thelung of a pig inoculated with 1-126 virus. No bronchiolar injuryresulted.

FIG. 7. Histopathology of TX/98/del 126-vaccinated pigs. FIG. 7 shows abar chart depicting the histopathological scores of lungs from pigsvaccinated with TX/98/del126. Mock=non vaccinated, sham-inoculated pigs;H3N2=non vaccinated pigs inoculated with 2×10⁵ PFU per pig withA/Swine/Texas/4199-2/98; H1N1=non-vaccinated pigs inoculated with 2×10⁵PFU per pig with A/Swine/MN/37866/99; MLV+Mock=TX/98/del 126 vaccinated,sham inoculated pigs; MLV+H3N2=TX/98/del 126 vaccinated pigs inoculatedwith 2×10⁵ PFU per pig with A/Swine/Texas/4199-2/98; MLV+H1N1=TX/98/del126 vaccinated pigs inoculated with 2×10⁵ PFU per pig withA/Swine/MN/37866/99.

FIG. 8: Immunohistochemistry for SIV-antigen in TX/98/del 126-vaccinatedpigs. FIG. 8 shows a bar chart depicting the immunohistological scoresfor pigs vaccinated with TX/98/del126. Mock=non vaccinated,sham-inoculated pigs; H3N2=non vaccinated pigs inoculated with 2×10⁵ PFUper pig with A/Swine/Texas/4199-2/98; H1N1=non-vaccinated pigsinoculated with 2×10⁵ PFU per pig with A/Swine/MN/37866/99;MLV+Mock=TX/98/del 126 vaccinated, sham inoculated pigs;MLV+H3N2=TX/98/del 126 vaccinated pigs inoculated with 2×10⁵ PFU per pigwith A/Swine/Texas/4199-2/98; MLV+H1N1=TX/98/del 126 vaccinated pigsinoculated with 2×10⁵ PFU per pig with A/Swine/MN/37866/99.

FIG. 9: Virus titers in lung lavage of TX/98/del 126-immunized pigschallenged with H3N2 and H1N1 SIVs. FIG. 9 shows a bar chart depictingthe viral titers in lung lavage from pigs vaccinated with TX/98/del126.Mock=non vaccinated, sham-inoculated pigs; H3N2=non vaccinated pigsinoculated with 2×10⁵ PFU per pig with A/Swine/Texas/4199-2/98;H1N1=non-vaccinated pigs inoculated with 2×10⁵ PFU per pig withA/Swine/MN/37866/99; MLV+Mock=TX/98/del 126 vaccinated, sham inoculatedpigs; MLV+H3N2=TX/98/del 126 vaccinated pigs inoculated with 2×10⁵ PFUper pig with A/Swine/Texas/4199-2/98; MLV+H1N1=TX/98/del 126 vaccinatedpigs inoculated with 2×10⁵ PFU per pig with A/Swine/MN/37866/99. Thelimit of virus detection was 10^(1.5) TCID₅₀/ml.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides attenuated swine influenza viruses havingan impaired ability to antagonize the cellular interferon (IFN)response, methods for producing such attenuated swine influenza viruses,and the use of such viruses in vaccine and pharmaceutical formulations.Such viruses are capable of generating an immune response and creatingimmunity but not causing illness, or causing fewer and/or less severesymptoms, i.e., the viruses have decreased virulence. Therefore, theyare ideal candidates for live virus vaccines. Moreover, the attenuatedswine influenza viruses can induce a robust IFN response which has otherbiological consequences in vivo, affording protection against subsequentinfectious diseases and/or inducing antitumor responses. Therefore, theattenuated swine influenza viruses can be used pharmaceutically, for theprevention or treatment of other infectious diseases and/orIFN-treatable diseases.

The invention is based, in part, on the Applicants' discovery that swineinfluenza viruses engineered to contain or containing a deletion(s) inthe NS1 gene have impaired replication relative to wild-type swineinfluenza viruses as demonstrated by fewer lung lesions upon infectionof pigs, reduced viral titers in bronchoalveolar lavage fluid (BALF) andreduced detection of virus on nasal swabs. Surprisingly, and contrary toresults seen with human influenza virus in mouse models, the length ofthe NS1 protein does not correlate with the level of attenuation of theswine influenza virus. Applicants have discovered that with swineinfluenza, a mutant virus with the shortest NS1 protein is the leastattenuated. In other words, swine influenza viruses containing shorterdeletions in the NS1 gene exhibit a greater attenuation in vivo thanswine influenza viruses containing longer deletions in their NS1 gene,or wild-type swine influenza viruses. Applicants have further discoveredthat the recombinant swine influenza viruses are impaired in theirability to inhibit IFN production in vitro, and they do not replicate asefficiently as the parental recombinant strain in 10-day old embryonatedhen eggs, in MDCK cells, in PK-15 cells or in an in vivo pig model.While not intending to be bound to any theory or explanation for themechanism of action of the swine influenza virus NS1 deletion mutants invivo, the attenuated features of such viruses are presumably due totheir levels of NS1 protein expression, their ability to induce a robustcellular IFN response, and their impaired ability to antagonize such aresponse. However, the beneficial features of such viruses may not besolely attributable to their effect on the cellular interferon response.Indeed, alterations in other activities associated with NS1, such as,alteration of pre-mRNA splicing, inhibition of cellular mRNApolyadenylation, poly(A)-containing mRNA nucleocytoplasmic transport,and stimulation of viral protein synthesis, may contribute to thedesired attenuated phenotype achieved by the introduction of mutationsin the NS1 gene of swine influenza virus.

An attenuated swine influenza of the present invention comprises amutation in a swine influenza NS1 gene that diminishes the ability ofthe NS1 gene product to antagonize the cellular interferon response. Inone embodiment, an attenuated swine influenza virus of the inventioncomprises a genome comprising a mutation in a swine influenza virus NS1gene that diminishes the ability of the NS1 gene product to antagonize acellular interferon response, and permits the attenuated virus, at amultiplicity of infection (MOI) of between 0.0005 and 0.001, 0.001 and0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007, 0.001,0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5,5.0, 5.5, or 6.0, to grow to titers between approximately 1 toapproximately 100 fold, approximately 5 to approximately 80 fold,approximately 20 to approximately 80 fold, or approximately 40 toapproximately 80 fold, approximately 1 to approximately 10 fold,approximately 1 to approximately 5 fold, approximately 1 toapproximately 4 fold, approximately 1 to approximately 3 fold,approximately 1 to approximately 2 fold, or approximately 1, 2, 3, 4, 5,6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 fold lower than wild-type swine influenza virus in cells(e.g., cells of a human (e.g., PerC6, a producer cell line derived fromhuman embryonic retinoblasts transformed with the E1 region ofAdenovirus 5), mouse, chicken (e.g., chicken embryo fibroblasts), rat,birds, or pig (e.g., PK(D1) cells, PK(15) cells, PK13 cells, NSK cells,LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells,PK-2a/CL 13 or SJPL cells)), as determined approximately 2 to 10 days, 3to 7 days, 3 to 5 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 dayspost-infection when propagated under the same conditions. The titers ofattenuated and wild-type swine influenza viruses can be determinedutilizing any technique well-known in the art or described herein,(e.g., hemagglutination assays, plaque assays, egg infectious doses(EID50), tissue culture infectious doses (TCID50), etc.) and the virusescan be propagated under conditions described herein or well-known in theart (e.g., in pig cells, MDCK cells (e.g., in MEM, 10% v/v fetal calfserum (FCS), 1% penicillin/streptomycin at 37° C. in a 5% CO₂ humidifiedincubator) or embryonated chicken eggs (e.g., in a stationary incubatorat 37° C. with 55% relative humidity). Alternatively, the viruses can bepropagated in cells (e.g., in pig cells, MDCK cells, etc.) that aregrown in serum-free or serum reduced (e.g., TPCK trypsin) medium. In oneembodiment, the growth of an attenuated swine influenza virus of theinvention is compared to a particular standard or reference, e.g.,wild-type swine influenza virus A/Swine/Texas/4199-2/98.

In a specific embodiment, an attenuated swine influenza virus of theinvention comprises a genome comprising a mutation in a swine influenzavirus NS1 gene that diminishes the ability of the NS1 gene product toantagonize a cellular interferon response, and permits the attenuatedvirus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001,0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007,0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 toapproximately 100 fold, approximately 5 to approximately 80 fold,approximately 20 to approximately 80 fold, or approximately 40 toapproximately 80 fold, approximately 1 to approximately 10 fold,approximately 1 to approximately 5 fold, approximately 1 toapproximately 4 fold, approximately 1 to approximately 3 fold,approximately 1 to approximately 2 fold, or approximately 1, 2, 3, 4, 5,6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 fold lower than wild-type swine influenza virus in pigcells, as determined approximately 2 to 10 days, 3 to 7 days, 3 to 5days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 days post-infection when propagatedunder the same conditions. The titers of attenuated and wild-type swineinfluenza viruses can be determined utilizing any technique well-knownin the art or described herein, (e.g., hemagglutination assays, plaqueassays, egg infectious doses (EID50), tissue culture infectious doses(TCID50), etc.) and the viruses can be propagated under conditionsdescribed herein or well-known in the art (e.g., in pig cells, MDCKcells (e.g., in MEM, 10% v/v fetal calf serum (FCS), 1%penicillin/streptomycin at 37° C. in a 5% CO₂ humidified incubator) orembryonated chicken eggs (e.g., in a stationary incubator at 37° C. with55% relative humidity). Alternatively, the viruses can be propagated incells (e.g., in pig cells, MDCK cells, etc.) that are grown inserum-free or serum reduced (e.g., TPCK trypsin) medium.

An attenuated swine influenza virus having the desired phenotype canitself be used as the active ingredient in vaccine, pharmaceutical orimmunogenic formulations. Alternatively, the attenuated swine influenzavirus can be used as the vector or “backbone” of recombinantly producedvaccines or immunogenic formulations. To this end, the “reversegenetics” technique can be used to engineer mutations or introduceforeign epitopes into the attenuated swine influenza virus, which wouldserve as the “parental” strain. In this way, vaccines can be designedfor immunization against strain variants, or in the alternative, againstcompletely different infectious agents or disease antigens (e.g., tumorantigens). For example, the attenuated virus can be engineered toexpress neutralizing epitopes of other preselected strains.Alternatively, epitopes of other viruses can be built into theattenuated swine influenza virus. Alternatively, epitopes of non-viralinfectious pathogens (e.g., parasites, bacteria, fungi) can beengineered into the attenuated swine influenza virus.

The present invention provides methods for vaccinating a subjectcomprising administering the vaccine formulations of the invention. Inone embodiment, the present invention provides a method comprisingadministering to a subject an effective amount of a vaccine formulationof the invention. In certain embodiments, the dose of the vaccineformulation administered to the subject is between about 10² to about10⁸, about 10³ to about 10⁷, or about 10⁴ to about 5×10⁶ pfu. In otherembodiments, the dose of the vaccine formulation administered is 10²,5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷ or 10⁸pfu. In other embodiments, the dose of an immunogenic formulation of theinvention administered to a subject is 10² to about 10⁸, about 10³ toabout 10⁷, or about 10⁴ to about 5×10⁶ pfu. In yet other embodiments,the dose of an immunogenic formulation of the invention administered toa subject is 10², 5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶,10⁷, 5×10⁷ or 10⁸ pfu. In specific embodiments, the subject is a donkey,zebra, camel, dog, avian (e.g., a duck). In a preferred embodiment, thesubject is a pig.

The present invention provides immunogenic formulations comprising anattenuated swine influenza virus of the invention and methods forinducing an immune response for the treatment, management or preventionof a swine influenza virus infection or a condition or symptomassociated therewith, an infection other than a swine influenza virusinfection or a condition or symptom associated therewith, or a conditionin which the attenuated swine influenza viruses can be used as a vectorto induce an immune response to a particular antigen associated with thecondition, comprising administering to a subject an immunogenicformulation of the invention. In one embodiment, the present inventionprovides a method for inducing an immune response for the treatment,management or prevention of a swine influenza virus infection or acondition or symptom associated therewith, an infection other than aswine influenza virus infection or a condition or symptom associatedtherewith, or a condition in which the attenuated swine influenzaviruses can be used as a vector to induce an immune response to aparticular antigen associated with the condition, comprisingadministering to a subject an effective amount of an immunogenicformulation of the invention. In certain embodiments, the dose of theimmunogenic formulation administered to the subject is between about 10²to about 10⁸, about 10³ to about 10⁷, about 10⁴ to about 5×10⁶ pfu orabout 10⁴ to about 10⁷ pfu. In other embodiments, the dose of animmunogenic formulation of the invention administered to a subject is10², 5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷or 10⁸ pfu. In specific embodiments, the subject is a donkey, zebra,camel, dog, avian (e.g., a duck). In a preferred embodiment, the subjectis a pig.

The attenuated swine influenza viruses, which induce robust IFNresponses in subjects, may also be used in pharmaceutical formulationsfor the prophylaxis or treatment of other infections, or IFN-treatablediseases such as cancer. In this regard, the tropism of the attenuatedswine influenza virus can be altered to target the virus to a desiredtarget organ, tissue or cells in vivo or ex vivo. Using this approach,the IFN response can be induced locally, at the target site, thusavoiding or minimizing the side effects of systemic IFN treatments. Tothis end, the attenuated swine influenza virus can be engineered toexpress a ligand specific for a receptor of the target organ, tissue orcells.

The present invention provides methods for preventing, managing ortreating an infection or IFN-treatable disease in a subject, other thana swine influenza viral infection or IFN-treatable disease caused byswine influenza virus, comprising administering a pharmaceuticalformulation of the invention. In one embodiment, the present inventionprovides a method for preventing, managing or treating an infection orIFN-treatable disease in a subject, other than a swine influenza viralinfection or IFN-treatable disease caused by swine influenza virus,comprising administering an effective amount of a pharmaceuticalformulation of the invention. In certain embodiments, the dose of thepharmaceutical formulation administered to the subject is between about10² to about 10⁸, about 10³ to about 10⁷, about 10⁴ to about 5×10⁶ pfuor about 10⁴ to about 10¹² pfu. In other embodiments, the dose of thepharmaceutical formulation administered to the subject is 10², 5×10²,10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷, 10⁸, 5×10⁸,1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or 10¹² pfu. In specificembodiments, the subject is a donkey, zebra, camel, dog, avian (e.g., aduck). In a preferred embodiment, the subject is a pig.

The present invention provides methods for preventing, managing ortreating cancer in a subject comprising administering a pharmaceuticalcomposition of the invention. In one embodiment, the present inventionprovides a method for preventing, managing or treating cancer in asubject comprising administering an effective amount of a pharmaceuticalcomposition of the invention. In certain embodiments, the dose of thepharmaceutical formulation administered to the subject is between about10² to about 10⁸, about 10³ to about 10⁷, about 10⁴ to about 5×10⁶ pfuor about 10⁴ to about 10¹² pfu. In other embodiments, the dose of thepharmaceutical formulation administered to the subject is 10², 5×10²,10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷, 10⁸, 5×10⁸,1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or 10¹² pfu.

Applicants demonstrated that attenuated swine influenza viruses withimpaired interferon antagonist activity were shown to replicate in vivogenerating titers that are lower than than detected with wild-type swineinfluenza viruses, but that are sufficient to induce immunological andcytokine responses. Thus, mutations which diminish but do not abolishthe IFN antagonist activity of the swine influenza virus are preferredfor vaccine formulations. Such viruses can be selected for growth inboth conventional and non-conventional substrates, and for intermediatevirulence.

The present invention provides cells containing (alternatively,comprising) swine influenza viruses of the present invention. In aspecific embodiment, a cell contains/comprises a swine influenza virusthat is genetically engineered. In certain embodiments, the attenuatedswine influenza virus contained in the cell is engineered to encode anepitope derived from another virus or a tumor antigen. In accordancewith this embodiment, the genome of the attenuated swine influenza virusmay comprise at least one segment derived from a different virus. Anycell can be infected with, contain or comprise an attenuated swineinfluenza virus of the invention including, but not limited to, MDCKcells, PK cells, Vero cells, primary human umbilical vein endothelialcells (HUVEC), H292 human epithelial cell line, HeLa cells, and swineembryonic kidney cells RES. In a preferred embodiment, the cell is a pigcell or a pig cell line. In certain embodiments, the cell (e.g., a pigcell or pig cell line) is interferon deficient.

The invention encompasses the use of substrates such as cells, celllines and embryonated eggs, to propagate the attenuated swine influenzaviruses of the invention. In one embodiment, the invention providesmethods for vaccine production, immunogenic formulation production andpharmaceutical production comprising propagating in a substrate anattenuated swine influenza virus of the present invention and collectingprogeny virus, wherein the substrate is a cell, cell line or embryonatedegg. In certain embodiments, the substrate is an IFN deficient system.Exemplary IFN-deficient systems include, but are not limited to, youngembryonated eggs (e.g., 6 to 10 days old, 6 to 9 days old, 6 to 8 daysold or 6 to 7 days old embyronated eggs), IFN-deficient cell lines (suchas VERO cells or genetically engineered cell lines such as STAT1knockouts). Embryonated eggs or cell lines pretreated with compoundsthat inhibit the IFN system (including drugs, antibodies, antisense,ribozymes, etc.) can also be used as an IFN-deficient system. Further,eggs deficient in the IFN system, e.g., eggs produced by STAT1 negativebirds, especially fowl, including but not limited to transgenicchickens, ducks or turkeys, may be used as an IFN-deficient system.

5.1 Generation of Mutants with Altered IFN Antagonist Activity

Any mutant swine influenza virus or strain which has a decreased IFNantagonist activity can be selected and used in accordance with theinvention. In one embodiment, naturally occurring mutants or variants,or spontaneous swine influenza mutants can be selected that have animpaired ability to antagonize the cellular IFN response. In anotherembodiment, mutant swine influenza viruses can be generated by exposingthe virus to mutagens, such as ultraviolet irradiation or chemicalmutagens, or by multiple passages and/or passage in non-permissivehosts. Screening in a differential growth system can be used to selectfor those mutants having impaired IFN antagonist function. Since swineinfluenza virus A has a segmented genome, the attenuated phenotype canbe transferred to another strain having a desired antigen byreassortment, (i.e., by coinfection of the attenuated virus and thedesired strain, and selection for reassortants displaying bothphenotypes). In a specific embodiment, the swine influenza viruses ofthe invention are not naturally occurring viruses. In another specificembodiment, the swine influenza viruses of the invention are geneticallyengineered viruses. In yet another specific embodiment, a swineinfluenza virus with naturally occurring mutations in the NS1 gene arenot encompassed by the invention. In yet another specific embodiment,known swine influenza viruses with mutations in the NS1 gene are notencompassed by the invention. In specific embodiments, the swineinfluenza viruses of the invention contain all or a portion of the NS1gene derived from human influenza viruses.

Mutations can be engineered into swine influenza virus using “reversegenetics” approaches. In this way, natural or other mutations whichconfer the attenuated phenotype can be engineered into vaccine strains.For example, deletions, insertions or substitutions of the coding regionof the swine influenza virus gene for IFN antagonist activity (i.e., theNS1 of swine influenza virus) can be engineered. Deletions,substitutions or insertions in the non-coding region of the swineinfluenza virus gene responsible for IFN antagonist activity are alsocontemplated. To this end, mutations in the signals responsible for thetranscription, replication, polyadenylation and/or packaging of the generesponsible or the IFN-antagonist activity can be engineered. Suchmutations, for example to the promoter, could down-regulate theexpression of the swine influenza virus gene responsible for IFNantagonist activity. Mutations in the promoter can be made, for example,by promoter shuffling (e.g., of the influenza B virus promoter), or inthe noncoding regions of the NS1 gene. Mutations in swine influenzavirus genes which may regulate the expression of the swine influenzavirus gene responsible for IFN antagonist activity (i.e., the swineinfluenza virus NS1 gene) are also within the scope of viruses that canbe used in accordance with the invention.

The present invention also provides swine influenza viruses comprisinggenomes comprising mutations to the NS1 gene segment that may not resultin an altered IFN antagonist activity or an IFN-inducing phenotype butrather results in altered viral functions and an attenuated phenotype,e.g., altered inhibition of nuclear export of poly(A)-containing mRNA,altered inhibition of pre-mRNA splicing, altered inhibition of theactivation of PKR by sequestering of dsRNA, altered effect ontranslation of viral RNA and altered inhibition of polyadenylation ofhost mRNA (e.g., see Krug in Textbook of Influenza, Nicholson et al. Ed.1998, 82-92, and references cited therein).

The reverse genetics technique involves the preparation of syntheticrecombinant viral RNAs that contain the non-coding regions of the swineinfluenza virus RNA which are essential for the recognition by viralpolymerases and for packaging signals necessary to generate a maturevirion. The recombinant RNAs are synthesized from a recombinant DNAtemplate and reconstituted in vitro with purified viral polymerasecomplex to form recombinant ribonucleoproteins (RNPs) which can be usedto transfect cells. A more efficient transfection is achieved if theviral polymerase proteins are present during transcription of thesynthetic RNAs either in vitro or in vivo. The synthetic recombinantRNPs can be rescued into infectious virus particles. The foregoingtechniques are described in U.S. Pat. No. 5,166,057 issued Nov. 24,1992; in U.S. Pat. No. 5,854,037 issued Dec. 29, 1998; in EuropeanPatent Publication EP 0702085A1, published Feb. 20, 1996; in U.S. patentapplication Ser. No. 09/152,845; in International Patent PublicationsPCT WO97/12032 published Apr. 3, 1997; WO96/34625 published Nov. 7,1996; in European Patent Publication EP-A780475; WO 99/02657 publishedJan. 21, 1999; WO 98/53078 published Nov. 26, 1998; WO 98/02530published Jan. 22, 1998; WO 99/15672 published Apr. 1, 1999; WO 98/13501published Apr. 2, 1998; WO 97/06270 published Feb. 20, 1997; and EPO 780475A1 published Jun. 25, 1997, each of which is incorporated byreference herein in its entirety.

The helper-free plasmid technology can also be utilized to engineer anattenuated swine influenza virus. For a description of helper-freeplasmid technology see, e.g., International Publication No. WO 01/04333;U.S. Pat. No. 6,649,372; Fodor et al., 1999, J. Virol. 73:9679-9682;Hoffmann et al., 2000, Proc. Natl. Acad. Sci. USA 97:6108-6113; andNeumann et al., 1999, Proc. Natl. Acad. Sci. USA 96:9345-9350, which areincorporated herein by reference in their entireties.

Attenuated viruses generated by the reverse genetics approach orhelper-free plasmid technology can be used in the vaccine, immunogenicand pharmaceutical formulations described herein. Reverse geneticstechniques or helper-free plasmid technology can also be used toengineer additional mutations to other viral genes important forvaccine, immunogenic and pharmaceutical formulation production—i.e., theepitopes of useful vaccine strain variants can be engineered into theattenuated swine influenza virus. Alternatively, completely foreignepitopes, including antigens derived from other viral or non-viralpathogens can be engineered into the attenuated strain. For example,antigens of non-related viruses, parasite antigens, bacterial or fungalantigens or tumor antigens can be engineered into the attenuated strain(e.g., epitopes of porcine reproductive and respiratory syndrome virus,porcine cytomegalo virus, porcine respiratory corona virus, porcineencephalomyocarditis virus, porcine epidemic diarrhea and antigenicdeterminants of non-viral swine pathogens such as bacteria, including,but not limited to, Brucella suis, and parasites, including, but notlimited to, roundworms (Ascaris suum), whipworms (Trichuris suis), or atumor antigen such as carcinoembryonic antigen (CEA), breast cancerantigen such as EGFR (Epidermal growth factor receptor), HER2 antigen(p185^(HER2)), HER2 neu epitope, cancer antigen-50 (CA-50), cancerantigen 15-3 (CA15-3) associated with breast cancer, carcinomaassociated antigen (CAA), melanoma antigen, and melanoma associatedantigens 100, 25, and 150). Alternatively, epitopes which alter thetropism of the virus in vivo can be engineered into the chimericattenuated viruses of the invention.

In a specific embodiment, a combination of reverse genetics techniquesor helper-free technology and reassortant techniques can be used toengineer attenuated viruses having the desired epitopes in swineinfluenza viruses. For example, an attenuated swine influenza virus(generated by, e.g., reverse genetics techniques, helper-free plasmidtechnology or a combination thereof) and a strain carrying the desiredvaccine epitope (generated by, e.g., natural selection, mutagenesis, byreverse genetics techniques, helper-free plasmid technology or acombination thereof) can be co-infected in hosts that permitreassortment of the segmented genomes. Reassortants that display boththe attenuated phenotype and the desired epitope can then be selected.In a particular embodiment, reassortment techniques can be used totransfer the attenuated phenotype from a parental swine influenza virusstrain (a natural mutant, a mutagenized virus, or a geneticallyengineered virus) to a different virus strain (a wild-type virus, anatural mutant, a mutagenized virus, or a genetically engineered virus).

In a specific embodiment, the present invention provides an attenuatedswine influenza virus comprising a genome comprising a mutation in theswine influenza virus NS1 gene that diminishes the ability of the NS1gene product to antagonize a cellular interferon response. In accordancewith this embodiment, the attenuated swine influenza virus is preferablygenetically engineered.

The attenuated swine influenza viruses of the invention may have one ormore or a combination of any of the following characteristics: theability to induce interferon activity at a level less than (e.g., 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 98% less than) that of wild-type swine influenzavirus, e.g., A/Swine/Texas/4199-2/98, as measured by standard interferonassays; a viral titer 1 to 50, 2 to 25, 2 to 10 or 3 to 7 fold lowerthan wild-type swine influenza virus, e.g., A/Swine/Texas/4199-2/98,when grown under the same conditions (e.g., inoculated at a MOI of0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 or 10 and grownin PK cells, PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13cells or SJPL cells and assayed in MDCK cells); a viral titer 1 to 10fold, 1 to 5 fold, 5-10 fold, 10 to 500, 20 to 250, 20 to 100 or 40 to80 fold lower than wild-type swine influenza virus, e.g.,A/Swine/Texas/4199-2/98, when isolated from BALF of infected pigs asassayed in a plaque assay performed on MDCK cells; or a reduced abilityto cause lung lesions in infected pigs.

In a specific embodiment, an attenuated swine influenza virus of theinvention comprises a genome comprising a mutation in a swine influenzavirus NS1 gene that diminishes the ability of the NS1 gene product toantagonize a cellular interferon response, and permits the attenuatedvirus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001,0.001 and 0.01, 0.01 and 0.1, 0.1 and 1, or 0.1 and 1, or a MOI of0.0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers betweenapproximately 1 to approximately 100 fold, approximately 5 toapproximately 80 fold, approximately 20 to approximately 80 fold, orapproximately 40 to approximately 80 fold, approximately 1 toapproximately 10 fold, approximately 1 to approximately 5 fold,approximately 1 to approximately 4 fold, approximately 1 toapproximately 3 fold, approximately 1 to approximately 2 fold, orapproximately 1, 2, 3, 4, 5, 6, 7, 8, or approximately 1, 2, 3, 4, 5, 7,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95or 100 fold lower than wild-type swine influenza virus in cells (e.g.,cells of a human (e.g., PerC6, a producer cell line derived from humanembryonic retinoblasts transformed with the E1 region of Adenovirus 5),mouse, chicken (e.g., chicken embryo fibroblasts), rat, birds, or pig(e.g., PK(D1) cells, PK(15) cells, PK13 cells, NSK cells, LLC-PK1 cells,LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 or SJPLcells)) as determined by a hemagglutination assay of BALF obtained frompigs or supernatants of pig cells approximately 2 to 10 days, 3 to 7days, 3 to 5 days or 2, 3, 5, 6, 7, 8, 9, 10 days post-infection or whenthe viruses are plagued on MDCK cells. In one embodiment, the growth ofan attenuated swine influenza virus of the invention is compared to aparticular standard or reference, e.g., wild-type swine influenza virusA/Swine/Texas/4199-2/98. In accordance with these embodiments, theattenuated virus may be genetically engineered to contain or expressnon-swine influenza virus nucleic acid sequences such, e.g., an epitopeof a foreign pathogen or a tumor antigen. Preferably, the non-swineinfluenza virus sequences do not include a nucleic acid sequence thatalters the attenuated phenotype of the virus. Accordingly, nucleic acidsequences encoding proteins, polypeptides or peptides with interferonantagonizing activity are preferably not engineered into swine influenzavirus.

The invention provides attenuated swine influenza viruses comprising agenome comprising at least two, at least three, at least four or moremutations in two, three, four or more swine influenza virus genes,wherein at least one of the mutations is in the NS1 gene and contributesto or is responsible (directly or indirectly) for the attenuation of thevirus and/or the diminished ability of the virus to antagonize acellular interferon response. In a specific embodiment, an attenuatedswine influenza virus of the invention comprises a genome comprising atleast two, at least three, at least four or more mutations in two,three, four or more swine influenza virus genes, wherein at least one ofthe mutations is in the NS1 gene and is responsible for the diminishedability of the NS1 gene product to antagonize a cellular interferonresponse, and permits the attenuated virus, at a MOI of between 0.0005and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or MOI of 0.0005,0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately1 to approximately 100 fold, approximately 5 to approximately 80 fold,approximately 20 to approximately 80 fold, or approximately 40 toapproximately 80 fold, approximately 1 to approximately 10 fold,approximately 1 to approximately 5 fold, approximately 1 toapproximately 4 fold, approximately 1 to approximately 3 fold,approximately 1 to approximately 2 fold, or approximately 1, 2, 3, 4, 5,6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 fold lower than wild-type swine influenza virus, e.g.,A/Swine/Texas/4199-2/98, in pig cells, as determined by, e.g.,hemagglutination assays, approximately 2 to 10 days, 3 to 7 days, or 2,3, 5, 6, 7, 8, 9, 10 days post-infection when the viruses are propagatedunder the same conditions. In another embodiment, an attenuated swineinfluenza virus of the invention comprises a genome comprising at leasttwo, three, four or more mutations in two, three, four or more swineinfluenza virus genes, wherein at least one of the mutations is in theNS1 gene and is responsible for the attenuation of the virus, andpermits the attenuated virus, at a MOI of between 0.0005 and 0.001,0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005, 0.0007,0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, or 6.0, to grow to titers between approximately 1 toapproximately 100 fold, approximately 5 to approximately 80 fold,approximately 20 to approximately 80 fold, or approximately 40 toapproximately 80 fold, approximately 1 to approximately 10 fold,approximately 1 to approximately 5 fold, approximately 1 toapproximately 4 fold, approximately 1 to approximately 3 fold,approximately 1 to approximately 2 fold, or approximately 1, 2, 3, 4, 5,6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 fold lower than wild-type swine influenza virus, e.g.,A/Swine/Texas/4199-2/98, in pig cells, as determined by, e.g.,hemagglutination assays, approximately 2 to 10 days, 3 to 7 days, 3 to 5days, or 2, 3, 5, 6, 7, 8, 9, 10 days post-infection when the virusesare propagated under the same conditions (e.g., in MDCK cells). Inaccordance with these embodiments, the attenuated virus have an impairedinterferon antagonist phenotype and the viruses may be geneticallyengineered to contain or express non-swine influenza virus nucleic acidsequences, such as, e.g., an epitope of a foreign pathogen (e.g.,epitopes of porcine reproductive and respiratory syndrome virus, porcinecytomegalo virus, porcine respiratory corona virus, porcineencephalomyocarditis virus, porcine epidemic diarrhea and antigenicdeterminants of non-viral pathogens such as bacteria and parasites, toname but a few) or a tumor antigen. Preferably, the non-swine influenzavirus sequences (heterologous sequences) do not include a nucleic acidsequence that alters the attenuated phenotype of the virus. Accordingly,nucleic acid sequences encoding proteins, polypeptides or peptides withinterferon antagonizing activity are preferably not engineered into theswine influenza virus.

Any mutation that results in the desired phenotype (preferably, animpaired ability to antagonize a cellular interferon response) can beintroduced into the swine influenza virus NS1 gene. Examples of thetypes of mutations that can be included in or introduced into swineinfluenza virus NS1 gene include, but are not limited to, deletions,substitutions, insertions and combinations thereof. One or moremutations can be located anywhere throughout the NS1 gene (e.g., theamino-terminus, the carboxy-terminus or somewhere in between) and/or theregulatory element of the NS1 gene. In a specific embodiment, anattenuated swine influenza virus of the invention comprises a genomecomprising a swine influenza virus NS1 gene with a mutation (e.g., adeletion or substitution) at the amino-terminus. In a preferredembodiment, an attenuated swine influenza virus of the inventioncomprises a genome comprising a swine influenza virus with a mutation(e.g., a deletion or substitution, preferably a deletion) at thecarboxy-terminus. In another embodiment, an attenuated swine influenzavirus of the invention comprises a genome comprising a mutation in aswine influenza virus NS1 gene resulting in a deletion consisting of 5,preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75,80, 85, 90, 95, 99, 100, 105, 110, 115, 120, 125, 126, 130, 135, 140,145, 150, 155, 160, 165, 170 or 175 amino acid residues from the carboxyterminus of NS1, or a deletion of between 5-170, 25-170, 50-170,100-170, 100-160 or 105-160 amino acid residues from thecarboxy-terminus. In another embodiment, an attenuated swine influenzavirus of the invention comprises a genome comprising a mutation in aswine influenza virus NS1 gene resulting in a deletion of all amino acidresidues except amino acid residues 1-126, amino acid residues 1-120,amino acid residues 1-115, amino acid residues 1-110, amino acidresidues 1-100, amino acid residues 1-99, amino acid residues 1-95,amino acid residues 1-85, amino acid residues 1-80, amino acid residues1-75, amino acid residues 1-73, amino acid residues 1-70, amino acidresidues 1-65 or amino acid residues 1-60, wherein the amino terminalamino acid is number 1. In accordance with these embodiments, theattenuated swine influenza virus is preferably genetically engineered.In a preferred embodiment, an attenuated swine influenza virus of theinvention is TX/98/del 126, TX/98/del 99 or TX/98/del 73. In a morepreferred embodiment, the attenuated swine influenza virus is TX/98/del126. In a still more preferred embodiment, the attenuated swineinfluenza virus is TX/98/del 99.

The attenuated swine influenza virus of the present invention may be achimeric virus that expresses a heterologous sequence e.g., antigens ofother vaccine strains (e.g., using reverse genetics, reassortment orhelper-free plasmid technology). Alternatively, the attenuated influenzaviruses may be engineered, using reverse genetics, reassortment orhelper-free plasmid technology with genetically engineered viruses, toexpress completely foreign epitopes, e.g., antigens of other infectiouspathogens, tumor antigens, or targeting antigens. In certainembodiments, the attenuated swine influenza viruses express aheterologous sequence derived from other swine infectious agents,non-swine infectious agents, swine or other types of tumor antigens(e.g., carcinoembryonic antigen (CEA), breast cancer antigen such asEGFR (epidermal growth factor receptor), HER2 antigen (p185HER2), HER2neu epitope, cancer antigen-50 (CA-50), cancer antigen 15-3 (CA15-3)associated with breast cancer, carcinoma associated antigen (CAA),melanoma antigen, and melanoma associated antigens 100, 25, and 150). Inother embodiments, the attenuated swine influenza virus of the presentinvention may contain a segment derived from another virus. Since the NSRNA segment is the shortest among the eight viral RNAs, it is possiblethat the NS RNA will tolerate longer insertions of heterologoussequences than other viral genes. Moreover, the NS RNA segment directsthe synthesis of high levels of protein in infected cells, suggestingthat it would be an ideal segment for insertions of foreign antigens.Exemplary sequences include epitopes of porcine reproductive andrespiratory syndrome virus, porcine cytomegalo virus, porcinerespiratory corona virus, porcine encephalomyocarditis virus, porcineepidemic diarrhea and antigenic determinants of non-viral swinepathogens such as bacteria, including, but not limited to, Brucellasuis, and parasites, including, but not limited to, roundworms (Ascarissuum), whipworms (Trichuris suis).

Preferably, the heterologous sequence is not a nucleic acid sequencethat alters the attenuated phenotype of the virus. Accordingly,preferably, nucleic acid sequences encoding proteins, polypeptides orpeptides with interferon antagonizing activity are not engineered intothe swine influenza virus. In certain embodiments, the chimeric virusexpresses a tumor antigen. In other embodiments, the chimeric virusexpresses an epitope of a foreign pathogen.

5.2 Selection of Attenuated Swine Influenza Viruses

The invention encompasses methods of selecting swine influenza viruseswhich have the desired phenotype, i.e., attenuated swine influenzaviruses or swine influenza viruses which have low IFN activity (i.e., areduction of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%,70-80%, 80-90% or more when compared to wild-type swine influenzaviruses, e.g., A/Swine/Texas/4199-2/98, under the same conditions) or noIFN antagonist activity whether obtained from natural variants,spontaneous variants (i.e., variants which evolve during viruspropagation), mutagenized natural variants, reassortants and/orgenetically engineered viruses (see, e.g., U.S. Pat. No. 6,635,416).Such viruses can be best screened in differential growth assays thatcompare growth in IFN-deficient versus IFN-competent host systems.Viruses which demonstrate better growth in the IFN-deficient systemsversus IFN competent systems are selected; preferably, viruses whichgrow to titers at least one log, at least two logs, three logs, or 1 to50, 1 to 25, 1 to 20, 1 to 10 or 1 to 5 logs greater in IFN-deficientsystems as compared to an IFN-competent system are selected. ExemplaryIFN-deficient systems include, but are not limited to, young embryonatedeggs (e.g., 6 to 10 days old, 6 to 9 days old, 6 to 8 days old or 6 to 7days old embryonated eggs), IFN-deficient cell lines (such as VERO cellsor genetically engineered cell lines such as STAT1 knockouts, PKRknockouts, etc.). Embryonated eggs or cell lines pretreated withcompounds that inhibit the IFN system (including drugs, antibodies,antisense, ribozymes, etc.) can also be used as an IFN-deficient system.Further, eggs deficient in the IFN system, e.g., eggs produced by STAT1negative birds, especially fowl, including but not limited to transgenicchickens, ducks or turkeys, may be used as an IFN-deficient system.Attenuated swine influenza viruses showing at least 1 to 50, 1 to 25, 1to 20, 1 to 10 or 1 to 5 logs lower titers in 10-days-old eggs versus6-7, 6-8, or 6-9 days old eggs will be considered impaired in theirability to inhibit the IFN response.

For purposes of screening, transient IFN-deficient systems can be usedin lieu of genetically manipulated systems. For example, the host systemcan be treated with compounds that inhibit IFN production and/orcomponents of the IFN response (e.g., drugs, antibodies against IFN,antibodies against IFN-receptor, inhibitors of PKR, antisense moleculesand ribozymes, etc.). Growth of attenuated swine influenza virus can becompared in IFN-competent untreated controls versus IFN-deficienttreated systems.

Growth of swine influenza virus (as measured by titer) can, e.g., becompared in a variety of cells, cell lines, or animal model systems thatexpress IFN and the components of the IFN response, versus cells, celllines, or animal model systems deficient for IFN or components of theIFN response. Techniques which are well known in the art for thepropagation of viruses in cell lines can be used (see, for example, theworking examples infra). Growth of swine influenza virus in an IFNcompetent cell line versus an IFN deficient genetically engineered cellline can be compared.

The swine influenza viruses can be screened using IFN assay systemse.g., transcription based assay systems in which reporter geneexpression is controlled by an IFN-responsive promoter. Reporter geneexpression in infected versus uninfected cells can be measured toidentify viruses which efficiently induce an IFN response, but which areunable to antagonize the IFN response. For example, test cells can beengineered to transiently or constitutively express reporter genes suchas luciferase reporter gene or chloramphenicol transferase (CAT)reporter gene under the control of an interferon stimulated responseelement, such as the IFN-stimulated promoter of the ISG-54K gene(Bluyssen et al., 1994, Eur. J. Biochem. 220:395-402). Cells areinfected with the test swine influenza virus and the level of expressionof the reporter gene compared to that in uninfected cells or cellsinfected with wild-type swine influenza virus. An increase in the levelof expression of the reporter gene following infection with the testattenuated swine influenza virus would indicate that the test swineinfluenza virus is inducing an IFN response. Alternatively, theinduction of IFN responses may be determined by measuring IFN-dependenttranscriptional activation following infection with the test attenuatedswine influenza virus. The expression of genes known to be induced byIFN, e.g., Mx, PKR, 2-5-oligoadenylatesynthetase, majorhistocompatibility complex (MHC) class I, etc., can be analyzed bytechniques known to those of skill in the art (e.g., northern blots,western blots, PCR, etc.). The induction of IFN responses may also bedetermined by measuring the phosphorylated state of components of theIFN pathway following infection with a test swine influenza virus, e.g.,IRF-3, which is phosphorylated in response to double-stranded RNA. Inresponse to type I IFN, Jak1 kinase and TyK2 kinase, subunits of the IFNreceptor, STAT1, and STAT2 are rapidly tyrosine phosphorylated. Thus, inorder to determine whether the swine influenza virus induces IFNresponses, cells, such as 293 cells, are infected with the test mutantvirus and following infection, the cells are lysed. IFN pathwaycomponents, such as Jak1 kinase or TyK2 kinase, are immunoprecipitatedfrom the infected cell lysates, using specific polyclonal sera orantibodies, and the tyrosine phosphorylated state of the kinase isdetermined by immunoblot assays with an anti-phosphotyrosine antibody(e.g., see Krishnan et al. 1997, Eur. J. Biochem. 247: 298-305). Anenhanced phosphorylated state of any of the components of the IFNpathway following infection with the swine influenza virus wouldindicate induction of IFN responses by the swine influenza virus.

Further, the induction of IFN responses following infection with a testswine influenza virus may be determined by measuring the ability to bindspecific DNA sequences or the translocation of transcription factorsinduced in response to viral infection, e.g., IRF3, STAT1, STAT2, etc.In particular, STAT1 and STAT2 are phosphorylated and translocated fromthe cytoplasm to the nucleus in response to type I IFN. The ability tobind specific DNA sequences or the translocation of transcriptionfactors can be measured by techniques known to those of skill in theart, e.g., electromobility gel shift assays, cell staining, etc. Otherassays that can be used are described in U.S. patent application Ser.No. 09/829,711, herein incorporated by reference in its entirety. In apreferred embodiment, however, differential growth assays are used toselect viruses having the desired phenotype, since the host system used(IFN-competent versus IFN-deficient) applies the appropriate selectionpressure.

The attenuated swine influenza viruses of the present invention areoptimally screened in pig cells, including primary, secondary pig cellsand pig cell lines. Any pig cell which is capable of growing swineinfluenza virus can be used. Preferred pig cells include porcine kidneycell lines, porcine testis cells lines and porcine lung. Representativepig cells include, but are not limited to, PK(D1) cells, PK(15) cells,PK13 cells, NSK cells, LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, STcells, PT-K75 cells, PK-2a/CL 13 cells or SJPL cells.

The swine influenza viruses of the invention can be screened in pigcells by comparison to wild-type swine influenza viruses, e.g.,A/Swine/Texas/4199-2/98, under the same growth conditions. Attenuatedswine influenza viruses are selected based on characteristics such asslower growth rates, fewer lung lesions upon infection of pigs, reducedviral titers in bronchoalveolar lavage fluid (BALF) and reduceddetection of virus on nasal swabs when compared to wild-type swineinfluenza virus. Such attenuated swine influenza viruses have decreasedvirulence because they have a reduced ability to replicate in interferoncompetent systems compared to wild-type swine influenza viruses but cangenerate an immune response.

Pig cells can be infected with wild-type swine influenza viruses, e.g.,A/Swine/Texas/4199-2/98, and NS1 mutants at a specific MOI and viraltiters in the supernatant can be determined at specific timespost-infection. Infection of pig cells at MOIs from between 0.0005 and0.001, 0.001 and 0.01, 0.01 and 0.1, 0.1 and 1, or 1 and 10, or a MOI of0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 or 10 can be used. Inone embodiment, an MOI of 0.001 is used. Viral titers can be determinedfrom approximately 2 to 10 days, 3 to 7 days, 3 to 5 days or 2, 3, 4, 5,6, 7, 8, 9 or 10 days post-infection. In a specific embodiment, viraltiters are determined 5 days post-infection. After growing virus in pigcells, viral titers can be assessed in the supernatant. Any system formeasuring titers of influenza virus can be used. Representative systemsare described in Section 5.7. In a particular embodiment, viral titersin the supernatant are determined by plaguing various time points onMDCK cells.

The selection systems of the invention encompass measuring IFN inductionby determining whether an extract from the cell or egg infected with thetest attenuated swine influenza virus is capable of conferringprotective activity against viral infection. More specifically, groupsof 10-day old embryonated chicken eggs are infected with the test mutantswine influenza virus or the wild-type swine influenza virus.Approximately 15 to 20 hours post infection, the allantoic fluid isharvested and tested for IFN activity by determining the highestdilution with protective activity against swine influenza virusinfection in tissue culture cells.

The pathogenesis of mutant swine influenza viruses of the invention canalso be assessed in pigs in vivo. Useful assays include assessing lunglesions in lung lobes, nasal swabs and determination of viral titers inbronchoalveolar lavage fluid (BALF) by any method known in the art.

5.3 Propagation of Attenuated Swine Influenza Virus

The present invention provides methods for propagating attenuated swineinfluenza viruses in cells (e.g., pig cells), embryonated eggs, andanimals. The attenuated swine influenza viruses of the present inventioncan be propagated in any substrate that allows the virus to grow totiters that permit a use of the attenuated swine influenza virusdescribed herein. In a specific embodiment, the attenuated swineinfluenza viruses of the present invention are propagated in anysubstrate that allows the virus to grow to titers comparable to thosedetermined for wild type swine influenza virus strains in IFN-competentsubstrates. In another embodiment, the attenuated swine influenzaviruses of the invention are propagated in IFN-deficient substrates.Substrates which are useful for selection of the attenuated swineinfluenza viruses of the invention do not have to be used forpropagation and vice versa.

In accordance with the methods of the present invention, the attenuatedswine influenza viruses which may be grown in cells (e.g., pig cells),embryonated eggs, and animals are selected from naturally occurringstrains, variants or mutants, mutagenized virus, reassortants and/orgenetically engineered viruses. The methods of the present inventionencompass growing the attenuated swine influenza viruses, preferablyusing appropriate growth conditions (see e.g., growth conditions setforth in Section 6 below), and collecting the progeny virus.

In a specific embodiment, the attenuated swine influenza viruses of theinvention are propagated in pig cells. In accordance with thisembodiment, the pig cells may or may not be IFN-deficient or producelower levels of IFN. Preferred pig cells include porcine kidney celllines, porcine testis cell lines and porcine lung cell lines.Representative pig cells include, but are not limited to, PK(D1) cells,PK(15) cells, PK13 cells, SJPL cells, NSK cells, LLC-PK1 cells, LLC-PK1Acells, ESK-4 cells, ST cells, PT-K75 cells, and PK-2a/CL 13 cells. Inanother specific embodiment, the attenuated swine influenza viruses arepropagated in chicken cells, e.g., chicken embryo fibroblasts derivedfrom 6-day-old embryonated eggs.

In certain embodiments, the invention provides methods of propagatingthe attenuated swine influenza viruses of the invention in embryonatedeggs, e.g., from 6 to 14 days old. In other embodiments, young orimmature embryonated eggs can be used to propagate attenuated swineinfluenza viruses of the invention. In accordance with the presentinvention, immature embryonated eggs encompass eggs which are less thanten-day-old eggs, preferably six to nine day old eggs, six to eight dayold, six to seven day old eggs or six days old eggs. Immatureembryonated eggs of the present invention also encompass eggs whichartificially mimic immature eggs up to, but less than ten-day-old, as aresult of alterations to the growth conditions, e.g., changes inincubation temperatures; treating with drugs; or any other alterationwhich results in an egg with a retarded development, such that the IFNsystem is not fully developed as compared with ten-to twelve-day-oldeggs. The swine influenza viruses can be propagated in differentlocations of the embryonated egg, e.g., the allantoic cavity. In certainembodiments, the embryonated eggs are chick eggs.

The invention also encompasses methods and IFN-deficient substrates forthe growth and isolation of attenuated swine influenza viruses of thepresent invention. See, e.g., U.S. Pat. No. 6,573,079, which isexpressly incorporated by reference in its entirety. IFN-deficientsubstrates which can be used to support the growth of the attenuatedswine influenza viruses include, but are not limited to, naturallyoccurring cells, cell lines, embryonated eggs, and IFN-deficientsystems, e.g., Vero cells, young embryonated eggs; recombinant cells orcell lines that are engineered to be IFN deficient, e.g., IFN-deficientcell lines derived from STAT1 knockouts, IRF3 knockouts, IRF7 knockouts,PKR knockouts, etc.; embryonated eggs obtained from IFN deficient birds,especially fowl (e.g., chickens, ducks, turkeys) including flock thatare bred to be IFN-deficient or transgenic birds (e.g., STAT1knockouts). In certain embodiments, the IFN-deficient substrate is notVero cells and/or not a STAT1 deficient cell line.

The host system, cells, cell lines, eggs or animals can be geneticallyengineered to express transgenes encoding inhibitors of the IFN system,e.g., dominant-negative mutants, such as STAT1 lacking the DNA bindingdomain, antisense RNA, ribozymes, inhibitors of IFN production,inhibitors of IFN signaling, and/or inhibitors of antiviral genesinduced by IFN. It should be recognized that animals that are bred orgenetically engineered to be IFN deficient will be somewhatimmunocompromised, and should be maintained in a controlled, diseasefree environment. Thus, appropriate measures (including the use ofdietary antibiotics) should be taken to limit the risk of exposure toinfectious agents of transgenic IFN deficient animals, such as mice,flocks of breeding hens, ducks, turkeys etc. Alternatively, the hostsystem, e.g., cells, cell lines, eggs or animals can be treated with acompound which inhibits IFN production and/or the IFN pathway e.g.,drugs, antibodies, antisense molecules, ribozyme molecules targeting theSTAT1 gene, and/or antiviral genes induced by IFN.

The present invention encompasses methods of growing and isolating swineinfluenza viruses having altered IFN antagonist activity in cells andcell lines which naturally do not have an IFN pathway or have adeficient IFN pathway or have a deficiency in the IFN system e.g., lowlevels of IFN expression as compared to wild-type cells. In a particularembodiment, the present invention encompasses methods of growing theattenuated swine influenza viruses of the invention in chicken embryofibroblasts derived from 6-day-old embryonated eggs, or Vero cells, orIFN-compromised embryonated eggs (e.g., immature embryonated eggs suchas 6-, 7- or 8-day old embryonated eggs). In another embodiment, thepresent invention encompasses methods of growing the attenuated swineinfluenza viruses of the invention in cells where the cells are not Verocells.

The present invention provides methods of growing and isolating theswine influenza viruses of the invention from a genetically engineeredIFN deficient substrate. The present invention encompasses transgenicpigs and avians in which a gene essential to the IFN system is mutated,e.g., STAT1, which would lay eggs that are IFN deficient. The presentinvention further encompasses avian transgenics which expressdominant-negative transcription factors, e.g., STAT1 lacking the DNAbinding domain, ribozymes, antisense RNA, inhibitors of IFN production,inhibitors of IFN signaling, and inhibitors of antiviral genes inducedin response to IFN.

The invention provides recombinant cell lines or animals, in particularpigs and avians, in which one or more genes essential for IFN synthesis,the IFN pathway, and/or an antiviral gene induced by IFN, e.g.interferon receptor, STAT1, PKR, IRF3, IRF7, etc. has been mutated(e.g., disrupted, i.e., is a “knockout”). The recombinant animal can beany animal (such as a mouse, a pig or an avian, e.g., chicken, turkey,hen, duck, etc. (see, e.g., Sang, 1994, Trends Biotechnol. 12:415;Perry, et al., 1993, Transgenic Res. 2:125; Stern, C. D., 1996, Curr TopMicrobiol Immunol 212:195-206; and Shuman, 1991, Experientia 47:897 forreviews regarding the production of avian transgenics each of which isincorporated by reference herein in its entirety)). In a specificembodiment, the recombinant animal is a pig. Such a cell line or animalcan be generated by any method known in the art for disrupting a gene onthe chromosome of the cell or animal. Such techniques include, but arenot limited to pronuclear microinjection (Hoppe & Wagner, 1989, U.S.Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines(Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152);gene targeting in embryonic stem cells (Thompson et al., 1989, Cell56:313); electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803);and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717);etc. For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115:171, which is incorporated by referenceherein in its entirety.

In particular, a STAT1 knockout animal can be produced by promotinghomologous recombination between a STAT1 gene in its chromosome and anexogenous STAT1 gene that has been rendered biologically inactive(preferably by insertion of a heterologous sequence, e.g., an antibioticresistance gene). Homologous recombination methods for disrupting genesin the mouse genome are described, for example, in Capecchi (1989,Science 244:1288) and Mansour et al. (1988, Nature 336:348-352).

Briefly, all or a portion of a STAT1 genomic clone is isolated fromgenomic DNA from the same species as the knock-out cell or animal. TheSTAT1 genomic clone can be isolated by any method known in the art forisolation of genomic clones (e.g. by probing a genomic library with aprobe derived from a STAT1 sequence such as pig STAT1 (Genbank AccessionNos. AB116564 and NM_213769) and those sequences provided in see Merazet al. 1996, Cell 84: 431-442; Durbin et al. 1996, Cell 84: 443-450, andreferences cited therein). Once the genomic clone is isolated, all or aportion of the clone is introduced into a recombinant vector.Preferably, the portion of the clone introduced into the vector containsat least a portion of an exon of the STAT1 gene, i.e., contains a STAT1protein coding sequence. A sequence not homologous to the STAT1sequence, preferably a positive selectable marker, such as a geneencoding an antibiotic resistance gene, is then introduced into theSTAT1 gene exon. The selectable marker is preferably operably linked toa promoter, more preferably a constitutive promoter. The non-homologoussequence is introduced anywhere in the STAT1 coding sequence that willdisrupt STAT1 activity, e.g., at a position where point mutations orother mutations have been demonstrated to inactivate STAT1 proteinfunction. For example, but not by way of limitation, the non-homologoussequence can be inserted into the coding sequence for the portion of theSTAT1 protein containing all or a portion of the kinase domain (e.g.,the nucleotide sequence coding for at least 50, 100, 150, 200 or 250amino acids of the kinase domain).

The positive selectable marker is preferably a neomycin resistance gene(neo gene) or a hygromycin resistance gene (hygro gene). The promotermay be any promoter known in the art; by way of example the promoter maybe the phosphoglycerate kinase (PGK) promoter (Adra et al., 1987, Gene60:65-74), the PolII promoter (Soriano et al., 1991, Cell 64:693-701),or the MC1 promoter, which is a synthetic promoter designed forexpression in embryo-derived stem cells (Thomas & Capecchi, 1987, Cell51:503-512). Use of a selectable marker, such as an antibioticresistance gene, allows for the selection of cells that haveincorporated the targeting vector (for example, the expression of theneo gene product confers resistance to G418, and expression of the hygrogene product confers resistance to hygromycin).

In a preferred embodiment, a negative selectable marker for acounterselection step for homologous, as opposed to non-homologous,recombination of the vector is inserted outside of the STAT1 genomicclone insert. For example, such a negative selectable marker is the HSVthymidine kinase gene (HSV-tk), the expression of which makes cellssensitive to ganciclovir. The negative selectable marker is preferablyunder the control of a promoter such as, but not limited to the PGKpromoter, the PolII promoter or the MC1 promoter.

When homologous recombination occurs, the portions of the vector thatare homologous to the STAT1 gene, as well as the non-homologous insertwithin the STAT1 gene sequences, are incorporated into the STAT1 gene inthe chromosome, and the remainder of the vector is lost. Thus, since thenegative selectable marker is outside the region of homology with theSTAT1 gene, cells in which homologous recombination has occurred (ortheir progeny), will not contain the negative selectable marker. Forexample, if the negative selectable marker is the HSV-tk gene, the cellsin which homologous recombination has occurred will not expressthymidine kinase and will survive exposure to ganciclovir. Thisprocedure permits the selection of cells in which homologousrecombination has occurred, as compared to non-homologous recombinationin which it is likely that the negative selectable marker is alsoincorporated into the genome along with the STAT1 sequences and thepositive selectable marker. Thus, cells in which non-homologousrecombination has occurred would most likely express thymidine kinaseand be sensitive to ganciclovir.

Once the targeting vector is prepared, it is linearized with arestriction enzyme for which there is a unique site in the targetingvector, and the linearized vector is introduced into embryo-derived stem(ES) cells (Gossler et al., 1986, Proc. Natl. Acad. Sci. USA83:9065-9069) by any method known in the art, for example byelectroporation. If the targeting vector includes a positive selectablemarker and a negative, counterselectable marker, the ES cells in whichhomologous recombination has occurred can be selected by incubation inselective media. For example, if the selectable markers are the neoresistance gene and the HSV-tk gene, the cells are exposed to G418(e.g., approximately 300 μg/ml) and ganciclovir (e.g., approximately 2μM).

Any technique known in the art for genotyping, for example but notlimited to Southern blot analysis or the polymerase chain reaction, canbe used to confirm that the disrupted STAT1 sequences have homologouslyrecombined into the STAT1 gene in the genome of the ES cells. Becausethe restriction map of the STAT1 genomic clone is known and the sequenceof the STAT1 coding sequence is known (see Meraz et al. 1996, Cell84:431, Durbin et al. 1996, Cell 84:443-450, all references citedtherein), the size of a particular restriction fragment or a PCRamplification product generated from DNA from both the disrupted andnon-disrupted alleles can be determined. Thus, by assaying for arestriction fragment or PCR product, the size of which differs betweenthe disrupted and non-disrupted STAT1 gene, one can determine whetherhomologous recombination has occurred to disrupt the STAT1 gene.

The ES cells with the disrupted STAT1 locus can then be introduced intoblastocysts by microinjection and then the blastocysts can be implantedinto the uteri of pseudopregnant mice using routine techniques. Theanimal that develop from the implanted blastocysts are chimeric for thedisrupted allele. The chimeric males can be crossed to females, and thiscross can be designed such that germline transmission of the allele islinked to transmission of a certain coat color. The germlinetransmission of the allele can be confirmed by Southern blotting or PCRanalysis, as described above, of genomic DNA isolated from tissuesamples.

Any gene whose product is important for interferon regulation can beused. Other mutations in the interferon pathway which may be used inaccordance with the present invention include kinase deficient versionsof Jak1, TyK2 or transcription factors lacking DNA binding domainsSTAT1, and STAT2 (see, e.g., Krishnan et al., 1997, Eur. J. Biochem.247: 298-305).

For virus purification, the attenuated swine influenza virus is removedfrom cell culture and separated from cellular components, typically bywell known clarification procedures, e.g., such as gradientcentrifugation and column chromatography, and may be further isolated asdesired using procedures well known to those skilled in the art, e.g.,plaque assays.

5.4 Uses of Attenuated Viruses

The attenuated swine influenza viruses of the invention can be used asviral vectors for production of heterologous proteins as described inU.S. Pat. Nos. 6,635,416 and 6,635,416.

The attenuated swine influenza viruses of the invention can be used asviral vectors for production of heterologous proteins as described inU.S. Pat. No. 5,820,871, which is herein incorporated by reference inits entirety.

The attenuated swine influenza viruses of the invention can be used inscreening assays to identify viral proteins with interferon antagonizingfunction, for identifying viral proteins that have the ability tocomplement replication of an attenuated virus with impaired ability toantagonize cellular interferon responses and screening assays toidentify anti-viral agents which inhibit interferon antagonist activityand inhibit viral replication as described in U.S. Pat. No. 6,635,416,which is herein incorporated by reference in its entirety.

The attenuated swine influenza viruses of the invention can be used toproduce antibodies which can be used in diagnostic immunoassays, passiveimmunotherapy, and generation of antiidiotypic antibodies. For example,an attenuated swine influenza virus comprising a genome comprising amutation in the NS1 gene and a heterologous sequence (e.g., a tumorantigen) can be administered to a subject (e.g., a pig) to generateantibodies which can then be isolated and used in diagnostic assays,passive immunotherapy and generation of antiidiotypic antibodies. Thegenerated antibodies may be isolated by standard techniques known in theart (e.g., immunoaffinity chromatography, centrifugation, precipitation,etc.) and used in diagnostic immunoassays, passive immunotherapy andgeneration of antiidiotypic antibodies. The isolated antibodies beforebeing used in passive immunotherapy may be modified, e.g., theantibodies may be chimerized or humanized. See, e.g., U.S. Pat. Nos.4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645,WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO91/10741; each of which is incorporated herein by reference in itsentirety, for reviews on the generation of chimeric and humanizedantibodies.

The antibodies isolated from subjects administered an attenuated swineinfluenza virus of the invention may also be used to monitor treatmentand/or disease progression. Any immunoassay system known in the art maybe used for this purpose including but not limited to competitive andnoncompetitive assay systems using techniques such as radioimmunoassays,ELISA (enzyme-linked immunosorbent assays), “sandwich” immunoassays,precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays and immunoelectrophoresis assays, to name but a few.

The antibodies generated by the attenuated swine influenza viruses ofthe invention can also be used in the production of antiidiotypicantibody. The antiidiotypic antibody can then in turn be used forimmunization, in order to produce a subpopulation of antibodies thatbind the initial antigen of the pathogenic microorganism (Jerne, 1974,Ann. Immunol. (Paris) 125c:373; Jerne et al., 1982, EMBO J. 1:234).

In immunization procedures, the amount of immunogen to be used and theimmunization schedule will be determined by a physician skilled in theart and will be administered by reference to the immune response andantibody titers of the subject.

5.5 Vaccine Formulations/Immunogenic Formulations

The invention encompasses vaccine formulations and immunogenicformulations comprising an attenuated swine influenza virus having animpaired ability to antagonize the cellular IFN response (i.e., swineinfluenza viruses with mutations in the NS1 gene that impair the abilityof the virus to induce a cellular IFN response), and a suitableexcipient. The attenuated swine influenza virus used in the vaccineformulation or immunogenic formulation may be selected from naturallyoccurring mutants or variants, mutagenized viruses or geneticallyengineered viruses. In a preferred embodiment, the attenuated swineinfluenza virus is genetically engineered. In another preferredembodiment, the attenuated swine influenza virus is isolated orpurified. Attenuated strains of swine influenza viruses can also begenerated via reassortment techniques, helper-free plasmid technology orby using a combination of the reverse genetics approach or helper-freeplasmid technology and reassortment techniques. Naturally occurringvariants include viruses isolated from nature as well as spontaneousoccurring variants generated during virus propagation, having animpaired ability to antagonize the cellular IFN response. The attenuatedswine influenza virus can itself be used as the active ingredient in thevaccine formulation or immunogenic formulation. Alternatively, theattenuated swine influenza virus can be used as the vector or “backbone”of recombinantly produced vaccines or immunogenic formulations. To thisend, recombinant techniques such as reverse genetics (or, for segmentedviruses, combinations of the reverse genetics and reassortmenttechniques) may be used to engineer mutations or introduce foreignantigens into the attenuated swine influenza virus used in the vaccineformulation or immunogenic formulation. In this way, vaccines can bedesigned for immunization against strain variants, or in thealternative, against completely different infectious agents or diseaseantigens.

Virtually any heterologous gene sequence may be constructed into theattenuated swine influenza viruses of the invention for use in vaccinesor immunogenic formulations. Preferably, epitopes that induce aprotective immune response to any of a variety of pathogens, or antigensthat bind neutralizing antibodies may be expressed by or as part of theviruses. For example, heterologous gene sequences that can beconstructed into the viruses of the invention for use in vaccines andimmunogenic formulations include but are not limited to antigenicdeterminants of non-viral pathogens such as bacteria and parasites. Inanother embodiment, all or portions of immunoglobulin genes may beexpressed. For example, variable regions of anti-idiotypicimmunoglobulins that mimic such epitopes may be constructed into theviruses of the invention. In yet another embodiment, tumor associatedantigens may be expressed. Examples of tumor antigens include, but arenot limited to, KS 1/4 pan-carcinoma antigen, ovarian carcinoma antigen(CA125), prostatic acid phosphate, prostate specific antigen,melanoma-associated antigen p97, melanoma antigen gp75, high molecularweight melanoma antigen (HMW-MAA), prostate specific membrane antigen,carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen,milk fat globule antigen, colorectal tumor-associated antigens (such as:CEA, TAG-72, CO17-1A; GICA 19-9, CTA-1 and LEA), Burkitt's lymphomaantigen-38.13, CD19, B-lymphoma antigen-CD20, CD33, melanoma specificantigens (such as ganglioside GD2, ganglioside GD3, ganglioside GM2,ganglioside GM3), tumor-specific transplantation type of cell-surfaceantigen (TSTA) (such as virally-induced tumor antigens includingT-antigen DNA tumor viruses and Envelope antigens of RNA tumor viruses),oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumoroncofetal antigen, differentiation antigen (such as human lung carcinomaantigen L6 and L20), antigens of fibrosarcoma, leukemia T cellantigen-Gp37, neoglycoprotein, sphingolipids, breast cancer antigens(such as EGFR (Epidermal growth factor receptor), HER2 antigen(p185^(HER2)) and HER2 neu epitope), polymorphic epithelial mucin (PEM),malignant human lymphocyte antigen-APO-1, differentiation antigen (suchas I antigen found in fetal erythrocytes, primary endoderm, I antigenfound in adult erythrocytes, preimplantation embryos, I(Ma) found ingastric adenocarcinomas, M18, M39 found in breast epithelium, SSEA-1found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, D₁56-22 found incolorectal cancer, TRA-1-85 (blood group H), C14 found in colonicadenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastriccancer, Y hapten, Le^(y) found in embryonal carcinoma cells, TL5 (bloodgroup A), EGF receptor found in A431 cells, E₁ series (blood group B)found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells,gastric adenocarcinoma antigen, CO-514 (blood group Le^(a)) found inAdenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood groupLe^(b)), G49 found in EGF receptor of A431 cells, MH2 (blood groupALe^(b)/Le^(y)) found in colonic adenocarcinoma, 19.9 found in coloncancer, gastric cancer mucins, T₅A₇ found in myeloid cells, R₂₄ found inmelanoma, 4.2, G_(D3), D1.1, OFA-1, G_(M2), OFA-2, G_(D2), andM1:22:25:8 found in embryonal carcinoma cells, and SSEA-3 and SSEA-4found in 4 to 8-cell stage embryos), T cell receptor derived peptidefrom a Cutaneous T cell Lymphoma, C-reactive protein (CRP), cancerantigen-50 (CA-50), cancer antigen 15-3 (CA15-3) associated with breastcancer, cancer antigen-19 (CA-19) and cancer antigen-242 associated withgastrointestinal cancers, carcinoma associated antigen (CAA),chromogranin A, epithelial mucin antigen (MC5), human epitheliumspecific antigen (HEA), Lewis(a)antigen, melanoma antigen, melanomaassociated antigens 100, 25, and 150, mucin-like carcinoma-associatedantigen, multidrug resistance related protein (MRPm6), multidrugresistance related protein (MRP41), Neu oncogene protein (C-erbB-2),neuron specific enolase (NSE), P-glycoprotein (mdr1 gene product),multidrug-resistance-related antigen, p170, multidrug-resistance-relatedantigen, prostate specific antigen (PSA), CD56, and NCAM.

Either a live recombinant swine influenza virus vaccine or immunogenicformulation or an inactivated recombinant swine influenza virus vaccineor immunogenic formulation can be formulated. A swine influenza viruscan be inactivated by methods well known to those of skill in the art.Common methods use formalin and heat for inactivation. See, e.g., U.S.Pat. No. 6,635,246, which is herein incorporated by reference in itsentirety. Other methods include those described in U.S. Pat. Nos.5,891,705; 5,106,619 and 4,693,981, which are herein incorporated byreference in their entireties.

A live vaccine or immunogenic formulation may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant swine influenza virus vaccine formulations and immunogenicformulations may be accomplished using conventional methods involvingpropagation of the swine influenza virus in cell culture or in theallantois of the chick embryo followed by purification. Moreover, theattenuated swine influenza viruses can induce a robust IFN responsewhich has other biological consequences in vivo, affording protectionagainst subsequent infectious diseases and/or inducing antitumorresponses.

Vaccine formulations and immunogenic formulations may includegenetically engineered swine influenza viruses that have mutations inthe NS1 gene including but not limited to the truncated NS1 influenzamutants described in the working examples, infra. They may also beformulated using natural variants. When formulated as a live virusvaccine, a range of about 10² to 10⁸ can be used, preferably from about10³ to 10⁷, more preferably 10⁴ pfu to about 5×10⁶, and most preferablyfrom 10⁴ to 10⁷ pfu per dose should be used.

In certain embodiments, swine influenza virus contain mutations to theNS1 gene segment that may not result in an altered IFN antagonistactivity or an IFN-inducing phenotype but rather result in altered viralfunctions and an attenuated phenotype e.g., altered inhibition ofnuclear export of poly(A)-containing mRNA, altered inhibition ofpre-mRNA splicing, altered inhibition of the activation of PKR bysequestering of dsRNA, altered effect on translation of viral RNA andaltered inhibition of polyadenylation of host mRNA (e.g., see Krug inTextbook of Influenza, Nicholson et al. Ed. 1998, 82-92, and referencescited therein).

Many methods may be used to introduce the vaccine formulations andimmunogenic formulations described above, these include but are notlimited to intranasal, intratracheal, oral, intradermal, intramuscular,intraperitoneal, intravenous, and subcutaneous routes. It may bepreferable to introduce the swine influenza virus vaccine formulation orimmunogenic formulation via the natural route of infection of thepathogen for which the vaccine is designed, or via the natural route ofinfection of the wild-type virus. Where a live attenuated swineinfluenza virus vaccine preparation or immunogenic formulation is used,it may be preferable to introduce the formulation via the natural routeof infection for influenza virus. The ability of influenza virus toinduce a vigorous secretory and cellular immune response can be usedadvantageously. For example, infection of the respiratory tract byattenuated swine influenza viruses may induce a strong secretory immuneresponse, for example in the urogenital system, with concomitantprotection against a particular disease causing agent.

In a specific embodiment, it may be desirable to administer thecompositions of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, by injection, by means of a catheter, orby means of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. In one embodiment, administration can be by direct injectionat the site (or former site) or infection.

A vaccine or immunogenic formulation of the present invention,comprising 10² to 10⁸ pfu can be used, preferably from about 10³ to 10⁷,more preferably 10⁴ pfu to about 5×10⁶ pfu of attenuated swine influenzavirus with altered IFN antagonist activity, could be administered onceto a subject. Alternatively, a vaccine or immunogenic formulation of thepresent invention, comprising 10² to 10⁸ can be used, preferably fromabout 10³ to 10⁷, more preferably 10⁴ to about 5×10⁶ pfu of mutantviruses with altered IFN antagonist activity, could be administeredtwice, three times, four times, five times, six times, seven times,eight times, nine times, or ten times to a subject with an interval of 2to 6 months, 2 to 8 months, 2 to 10 months, or 2 to 12 months betweendoses. Alternatively, a vaccine or immunogenic formulation of thepresent invention, comprising 10² to 10⁸ can be used, preferably fromabout 10³ to 10⁷, more preferably 10⁴ pfu to about 5×10⁶ pfu of mutantviruses with altered IFN antagonist activity, could be administered asoften as needed to a subject. In a specific embodiment, the subject is apig.

In certain embodiments, a vaccine formulation or an immunogenicformulation of the invention does not result in complete protection froman infection (e.g., a viral infection), but results in a lower titer orreduced number of the pathogen (e.g., a virus) compared to an untreatedsubject. Benefits include, but are not limited to, less severity ofsymptoms of the infection and a reduction in the length of the diseaseor condition associated with a the infection

In certain embodiments, the vaccine formulation or immunogenicformulation of the invention is used to protect against infections byswine influenza virus. In other embodiments, a vaccine formulation ofthe invention is used to protect against infection by another swinevirus, including, but not limited to, porcine reproductive andrespiratory syndrome virus, porcine cytomegalo virus, porcinerespiratory corona virus, porcine encephalomyocarditis virus, porcineepidemic diarrhea. In yet other embodiments, the vaccine formulation orimmunogenic formulation of the invention is used to protect againstinfections by pathogens other than a swine virus.

In certain embodiments, an immunogenic formulation of the invention isused for preventing, treating, managing or ameliorating conditions inwhich the induction of an immune response to a particular antigen isbeneficial.

5.6 Pharmaceutical Formulations

The present invention encompasses pharmaceutical formulations comprisingswine influenza viruses with altered IFN antagonist activity to be usedas anti-viral agents or anti-tumor agents or as agents againstIFN-treatable diseases. In one embodiment, the pharmaceuticalformulations have utility as an anti-viral prophylactic and may beadministered to a pig at risk of getting infected or is expected to beexposed to a virus, e.g., swine influenza virus. In another embodiment,the pharmaceutical formulations have utility as a therapeutic orprophylatic for an IFN-treatable disease and thus, can be used fortreating, preventing, managing or ameliorating an IFN-treatabledisorders. In a preferred embodiment, the pharmaceutical formulationshave utility as a therapeutic or prophylatic for a swine IFN-treatabledisease and thus, can be used for treating, preventing, managing orameliorating a swine IFN-treatable disorders. In specific embodiments,the pharmaceutical formulations of the invention have utility as atherapeutic or prophylactic for an IFN-treatable disease and thus, canbe used for treating, preventing, managing or ameliorating anIFN-treatable disorders in donkeys, zebras, camels, dogs, avians (e.g.,ducks). In a preferred embodiment, the pharmaceutical formulations ofthe invention have utility as a therapeutic or prophylactic for anIFN-treatable disease and thus, can be used for treating, preventing,managing or ameliorating an IFN-treatable disorders in pigs.

In certain embodiments, a pharmaceutical formulation of the inventioncomprises a chimeric attenuated swine influenza virus comprising aheterologous sequence. The heterologous sequence can encode an epitopefrom a foreign or tumor antigen. A foreign antigen can be selected fromanother virus, a bacteria or a parasite. In one embodiment, thepharmaceutical formulations may be used to treat tumors or prevent tumorformation, e.g., in pigs who have cancer or in those who are at highrisk for developing neoplasms or cancer. For example, a subject (e.g., apig, donkey, or other animal that can be infected with swine influenzavirus or in which swine influenza virus can replicate) with cancer canbe treated to prevent further tumorigenesis. Alternatively, pigs who areor are expected to be exposed to carcinogens can be treated.Alternatively, pigs who are to be exposed to radiation can be treatedprior to exposure and thereafter. Specific cancers that can be treatedwith the methods and compostions of the present invention include, butare not limited to, cancers of the uterus, mammary gland, pancreas,skin, lymph gland, vulva and testicle.

The antitumor properties of the invention can be at least partiallyrelated to their ability to induce IFN and IFN responses. Alternatively,the antitumor properties of the attenuated swine influenza viruses ofthe invention can be related to their ability to specifically grow inand kill tumor cells, many of which are known to have deficiencies inthe IFN system. Regardless of the molecular mechanism(s) responsible forthe antitumor properties, the attenuated swine influenza viruses of theinvention may be used to treat tumors or to prevent tumor formation.

The present invention further encompasses the swine influenza viruseswith an altered IFN-antagonist phenotype which are targeted to specificorgans, tissues and/or cells in the body in order to induce therapeuticor prophylactic effects locally. One advantage of such an approach isthat the IFN-inducing swine influenza viruses of the invention aretargeted to specific sites, e.g. the location of a tumor, to induce IFNin a site specific manner for a therapeutic effect rather than inducingIFN systemically which may have toxic effects.

The IFN-inducing swine influenza viruses of the invention may beengineered using the methods described herein to express proteins orpeptides which would target the viruses to a particular site. In aspecific embodiment, the IFN-inducing swine influenza viruses would betargeted to sites of tumors. In such an embodiment, the swine influenzaviruses can be engineered to express the antigen combining site of anantibody which recognizes a tumor specific antigen, thus targeting theIFN-inducing swine influenza virus to the tumor. In another embodiment,where the tumor to be targeted expresses a hormone receptor, such asbreast or ovarian tumors which express estrogen receptors, theIFN-inducing swine influenza virus may be engineered to express theappropriate hormone. In yet another embodiment, where the tumor to betargeted expresses a receptor to a growth factor, e.g. VEGF, EGF, orPDGF, the IFN-inducing virus may be engineered to express theappropriate growth factor or fragment thereof. Thus, in accordance withthe invention, the IFN-inducing swine influenza viruses may beengineered to express any target gene product, including peptides,proteins, such as enzymes, hormones, growth factors, antigens orantibodies, which will function to target the swine influenza virus to asite in need of treatment (e.g., anti-viral, antibacterial,anti-microbial or anti-cancer therapy).

Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The pharmaceutical formulations of thepresent invention may be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. Administration can be systemic or local. In addition, ina preferred embodiment it may be desirable to introduce thepharmaceutical formulations of the invention into the lungs by anysuitable route. Pulmonary administration can also be employed, e.g., byuse of an inhaler or nebulizer, and formulation with an aerosolizingagent.

In a specific embodiment, it may be desirable to administer thepharmaceutical formulations of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

In another embodiment, the pharmaceutical formulation can be deliveredin a biological matrix. An example of a biological matrix is describedin U.S. Pat. No. 5,962,427, and U.S. Patent Application Publication U.S.2002/0193338, herein incorporated by reference in their entireties.

In yet another embodiment, the pharmaceutical formulation can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger & Peppas, 1983, J. Macromol. Sci.Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190;During et al., 1989, Ann. Neurol. 25:351 (1989); Howard et al., 1989, J.Neurosurg. 71:105). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, i.e., thelung, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, 1984, in Medical Applications of Controlled Release, supra,vol. 2, pp. 115-138). Other controlled release systems are discussed inthe review by Langer (1990, Science 249:1527-1533).

In a preferred embodiment, the pharmaceutical formulations of thepresent invention comprise an effective amount of an attenuated swineinfluenza virus of the invention, and a pharmaceutically acceptablecarrier. The term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopoeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the pharmaceuticalformulation is administered. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. These compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain an effective amount of the therapy, preferably in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the patient. The formulation shouldsuit the mode of administration. The particular formulation may alsodepend on whether swine influenza virus is live or inactivated.

The amount of the pharmaceutical formulation of the invention which willbe effective in the treatment, prevention, management, or ameliorationof a particular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.However, suitable dosage ranges for administration are generally about10², 5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷,10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or 10¹² pfu,and most preferably about 10⁴ to about 10¹² pfu, and can be administeredto a subject once, twice, three or more times with intervals as often asneeded. Pharmaceutical formulations of the present invention comprising10², 5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷,10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or 10¹² pfu,and most preferably about 10⁴ to about 10¹² pfu of an attenuated swineinfluenza virus with altered IFN antagonist activity, can beadministered to a subject intranasally, intratracheally, intramuscularlyor subcutaneously Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

In certain embodiments, the pharmaceutical formulation of the inventionis used to prevent, manage, treat or ameliorate infections by viruses,bacteria or parasites. In other embodiments, the pharmaceuticalformulation of the invention is used to prevent, manage, treat orameliorate cancer in a pig. In other embodiments, the pharmaceuticalformulation of the invention is used to prevent, manage, treat orameliorate other IFN-treatable diseases.

5.7 Therapies Useful in Combination with Swine Influenza Virus

The present invention also provides methods for preventing, managing,treating, and/or ameliorating diseases and disorders including, but notlimited to, swine influenza virus infections, conditions associated withswine influenza virus infection, infections other than swine influenzavirus infections, conditions associated with infections other than swineinfluenza virus infections, IFN-treatable disorders (e.g., cancer) andconditions in which an attenuated swine influenza virus of the inventionis used as a vector to induce an immune response to an antigenassociated with the condition comprising administering to a subject inneed thereof an effective amount of one or more attenuated swineinfluenza viruses of the present invention and an effective amount ofone or more therapies (e.g., prophylactic or therapeutic agents) otherthan attenuated swine influenza viruses. Therapies include, but are notlimited to, small molecules, synthetic drugs, peptides, polypeptides,proteins, nucleic acids (e.g., DNA and RNA nucleotides including, butnot limited to, antisense nucleotide sequences, triple helices, RNAi,and nucleotide sequences encoding biologically active proteins,polypeptides or peptides) antibodies, synthetic or natural inorganicmolecules, mimetic agents, and synthetic or natural organic molecules.

Any therapy (e.g., prophylactic or therapeutic agents) which is known tobe useful, or which has been used or is currently being used for theprevention, management, treatment, or amelioration of a swine influenzavirus infection, infections other than swine influenza virus infections,conditions associated with infections other than swine influenza virusinfections, IFN-treatable disorders, conditions in which an attenuatedswine influenza virus of the invention is used as a vector to induce animmune response to an antigen associated with the condition, or anyother pig disease can be used in combination with an attenuated swineinfluenza virus in accordance with the invention described herein. See,e.g., Taylor, Pig Diseases, 6^(th) Ed., Diamond Farm Book Pubns, 1995;Straw et al., Diseases of Swine, 8^(th) Ed., Iowa State UniversityPress, 1999, for information regarding therapies, in particularprophylactic or therapeutic agents, which have been or are currentlybeing used for preventing, treating, managing, and/or ameliorating pigdiseases. Examples of prophylactic and therapeutic agents include, butare not limited to, anti-cancer agents; anti-viral agents,anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids (e.g.,beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone,methlyprednisolone, prednisolone, prednisone, hydrocortisone)) andantibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,erythomycin, penicillin, mithramycin, and anthramycin (AMC)).

5.7.1 Anti-Cancer Therapies

Any therapy (e.g., therapeutic or prophylactic agent) which is known tobe useful, has been used, or is currently being used for the prevention,treatment, management, or amelioration of cancer or one or more symptomsthereof can be used in compositions and method of the invention.Therapies (e.g., therapeutic or prophylactic agents) include, but arenot limited to, peptides, polypeptides, fusion proteins, nucleic acidmolecules, small molecules, mimetic agents, synthetic drugs, inorganicmolecules, and organic molecules. Non-limiting examples of cancertherapies include chemotherapies, radiation therapies, hormonaltherapies, and/or biological therapies/immunotherapies.

In particular embodiments, the anti-cancer agent may be, but is notlimited to: a chemotherapeutic agent (e.g., acivicin, aclarubicin,acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine,ambomycin, ametantrone acetate, aminoglutethimide, amsacrine,anastrozole, anthramycin, asparaginase, azacitidine, azetepa,batimastat, bleomycin, benzodepa, bicalutamide, bisantrenehydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate,brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone,campathecin, caracemide, carbetimer, carboplatin, carmustine, carubicinhydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin,cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine,cyclosporin A, combretastatin A4, combretastatin analogue, cytolyticfactor, cytostatin, dacliximab, docetaxel, dacarbazine, dactinomycin,daunorubicin hydrochloride, docetaxel, doxorubicin, droloxifene,dromostanolone propionate, duazomycin, edatrexate, eflornithinehydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine,epirubicin hydrochloride, erbulozole, esorubicin hydrochloride,estramustine, etanidazole, etoposide, fazarabine, fenretinide,floxuridine, fluorouracil, flurocitabine, fosquidone, gemcitabine,hydroxyurea, idarubicin, idarubicin hydrochloride, ifosfamide,ilmofosine, iproplatin, ifosfamide, irinotecan hydrochloride, lanreotideacetate, letrozole, leuprolide acetate, liarozole hydrochloride,lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol,mercaptopurine, methotrexate, metoprine, meturedepa, mitindomide,mitocarcin, mitocromin, mitomycin, mitoxantrone, mycophenolic acid,nitrosoureas, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel,plicamycin, platinum complex, platinum compounds, platinum-triaminecomplex, procarbizine, puromycin, taxol, thioguanine, talisomycin,tecogalan sodium, tegafur, teloxantrone hydrochloride, vapreotide,verteporfin, vinblastine sulfate, vincristine sulfate, vindesine,vindesine sulfate, vinepidine sulfate, vinglycinate sulfate,vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate,vinzolidine sulfate, vorozole, zeniplatin, and zinostatin), matrixmetalloproteinase inhibitors, tyrosine kinase inhibitors, tyrphostins,urokinase receptor antagonists, multiple drug resistance gene inhibitor,multiple tumor suppressor 1-based therapy, plasminogen activatorinhibitor, bisphosphonates (e.g., pamidronate (Aredria), sodiumclondronate (Bonefos), zoledronic acid (Zometa), alendronate (Fosamax),etidronate, ibandornate, cimadronate, risedromate, and tiludromate), acytokine (e.g., IL-2, IFN-α, IFN-β, IFN-γ, leukemia inhibiting factor,leukocyte alpha interferon, human chorionic gonadotrophin, andthrombopoietin), a hormone (e.g., thyroid stimulating hormone), anantibody (e.g., an anti-CD2 antibody, an anti-CD20 antibody, and anantibody immunospecific for one or more galanin receptors), vitamin D(e.g., 20-epi-1,25 dihydroxyvitamin D3), an angiogenesis inhibitor, anantisense oligonucleotide, an apoptosis gene modulator, an apoptosisregulator, a BCR/ABL antagonist, a cartilage derived inhibitor, anestrogen agonist, an estrogen antagonist, a gelatinase inhibitor, aglutathione inhibitor, an HMG CoA reductase inhibitor (e.g.,atorvastatin, cerivastatin, fluvastatin, lescol, lupitor, lovastatin,rosuvastatin, and simvastatin), an immunostimulant peptide, aninsulin-like growth factor-1 receptor inhibitor, an interferon agonist,leuprolide+estrogen+progesterone, leuprorelin, a mismatched doublestranded RNA, a proteasome inhibitor, a protein A-based immunemodulator, a protein kinase C inhibitor, a protein tyrosine phosphataseinhibitor, a raf antagonist, a ras farnesyl protein transferaseinhibitor, a ribozyme, RNAi, a signal transduction modulator, a stemcell inhibitor, a stem-cell division inhibitor, a telomerase inhibitor,a thymopoietin receptor agonist, and a translation inhibitor.

In specific embodiments, radiation therapy comprising the use of x-rays,gamma rays and other sources of radiation to destroy the cancer cells isused in combination with the attenuated swine influenza viruses of theinvention. In preferred embodiments, the radiation treatment isadministered as external beam radiation or teletherapy, wherein theradiation is directed from a remote source. In other preferredembodiments, the radiation treatment is administered as internal therapyor brachytherapy wherein a radioactive source is placed inside the bodyclose to cancer cells or a tumor mass.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physicians' Desk Reference (58th ed., 2004) and MerckVeterinary Manual (8th ed., 1998).

5.7.2 Anti-Angiogenesis Therapies

Any anti-angiogenic agent well-known to one of skill in the art can beused in the compositions and methods of the invention. Non-limitingexamples of anti-angiogenic agents include proteins, polypeptides,peptides, fusion proteins, antibodies (e.g., chimeric, monoclonal,polyclonal, Fvs, ScFvs, Fab fragments, F(ab)₂ fragments, andantigen-binding fragments thereof) such as antibodies thatimmunospecifically bind to TNFα, nucleic acid molecules (e.g., antisensemolecules or triple helices), organic molecules, inorganic molecules,and small molecules that reduce or inhibit or neutralizes theangiogenesis. In particular, examples of anti-angiogenic agents,include, but are not limited to, endostatin, angiostatin, apomigren,anti-angiogenic antithrombin III, the 29 kDa N-terminal and a 40 kDaC-terminal proteolytic fragments of fibronectin, a uPA receptorantagonist, the 16 kDa proteolytic fragment of prolactin, the 7.8 kDaproteolytic fragment of platelet factor-4, the anti-angiogenic 24 aminoacid fragment of platelet factor-4, the anti-angiogenic factordesignated 13.40, the anti-angiogenic 22 amino acid peptide fragment ofthrombospondin I, the anti-angiogenic 20 amino acid peptide fragment ofSPARC, RGD and NGR containing peptides, the small anti-angiogenicpeptides of laminin, fibronectin, procollagen and EGF, integrin α_(V)β₃antagonists (e.g., anti-integrin α_(V)β₃ antibodies), acid fibroblastgrowth factor (aFGF) antagonists, basic fibroblast growth factor (bFGF)antagonists, vascular endothelial growth factor (VEGF) antagonists(e.g., anti-VEGF antibodies), and VEGF receptor (VEGFR) antagonists(e.g., anti-VEGFR antibodies).

5.7.3 Anti-Viral Therapies

Any anti-viral agent well-known to one of skill in the art can be usedin the compositions and the methods of the invention. Non-limitingexamples of anti-viral agents include proteins, polypeptides, peptides,fusion proteins antibodies, nucleic acid molecules, organic molecules,inorganic molecules, and small molecules that inhibit and/or reduce theattachment of a virus to its receptor, the internalization of a virusinto a cell, the replication of a virus, or release of virus from acell. In particular, anti-viral agents include, but are not limited to,nucleoside analogs (e.g., zidovudine, acyclovir, gangcyclovir,vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet,amantadine, rimantadine, saquinavir, indinavir, ritonavir,alpha-interferons and other interferons, and AZT.

In specific embodiments, the anti-viral agent is an immunomodulatoryagent that is immunospecific for a viral antigen. As used herein, theterm “viral antigen” includes, but is not limited to, any viral peptide,polypeptide and protein (e.g., 3AB, 3ABC of foot-and-mouth diseasevirus, GP5 of porcine reproductive and respiratory syndrome virus,pseudorabies virus gE, swine transmissible gastroenteritis virusnucleoprotein, influenza virus neuraminidase, influenza virushemagglutinin, herpes simplex virus glycoprotein (e.g., gB, gC, gD, andgE) and hepatitis B surface antigen) that is capable of eliciting animmune response. Antibodies useful in this invention for treatment of aviral infectious disease include, but are not limited to, antibodiesagainst antigens of pathogenic viruses, including as examples and not bylimitation: adenovirdiae (e.g., mastadenovirus and aviadenovirus),herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus 2,herpes simplex virus 5, and herpes simplex virus 6), leviviridae (e.g.,levivirus, enterobacteria phase MS2, allolevirus), poxviridae (e.g.,chordopoxvirinae, parapoxvirus, avipoxvirus, capripoxvirus,leporiipoxvirus, suipoxvirus, molluscipoxvirus, and entomopoxvirinae),papovaviridae (e.g., polyomavirus and papillomavirus), paramyxoviridae(e.g., paramyxovirus, parainfluenza virus 1, mobillivirus (e.g., measlesvirus), rubulavirus (e.g., mumps virus), pneumonovirinae (e.g.,pneumovirus, human respiratory synctial virus), and metapneumovirus(e.g., avian pneumovirus and human metapneumovirus)), picornaviridae(e.g., enterovirus, rhinovirus, hepatovirus (e.g., human hepatits Avirus), cardiovirus, and apthovirus), reoviridae (e.g., orthoreovirus,orbivirus, rotavirus, cypovirus, fijivirus, phytoreovirus, andoryzavirus), retroviridae (e.g., mammalian type B retroviruses,mammalian type C retroviruses, avian type C retroviruses, type Dretrovirus group, BLV-HTLV retroviruses, lentivirus (e.g., humanimmunodeficiency virus 1 and human immunodeficiency virus 2),spumavirus), flaviviridae (e.g., hepatitis C virus), hepadnaviridae(e.g., hepatitis B virus), togaviridae (e.g., alphavirus (e.g., sindbisvirus) and rubivirus (e.g., rubella virus)), rhabdoviridae (e.g.,vesiculovirus, lyssavirus, ephemerovirus, cytorhabdovirus, andnecleorhabdovirus), arenaviridae (e.g., arenavirus, lymphocyticchoriomeningitis virus, Ippy virus, and lassa virus), and coronaviridae(e.g., coronavirus and torovirus).

Specific examples of antibodies available useful for the treatment of aviral infectious disease include, but are not limited to, PRO542(Progenics) which is a CD4 fusion antibody useful for the treatment ofHIV infection; Ostavir (Protein Design Labs, Inc., CA) which is a humanantibody useful for the treatment of hepatitis B virus; and Protovir(Protein Design Labs, Inc., CA) which is a humanized IgG1 antibodyuseful for the treatment of cytomegalovirus (CMV).

Anti-viral therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physicians' Desk Reference (58th ed., 2004) and MerckVeterinary Manual (8th ed., 1998). Additional information on viralinfections is available in Cecil Textbook of Medicine (18th ed., 1988).

5.7.4 Antibiotic Therapies

Any antibiotic agent well-known to one of skill in the art can be usedin the compositions and the methods of the invention. Antibacterialagents or antibiotics that can be used in combination with the complexesof the invention include but are not limited to: aminoglycosideantibiotics (e.g., apramycin, arbekacin, bambermycins, butirosin,dibekacin, neomycin, neomycin, undecylenate, netilmicin, paromomycin,ribostamycin, sisomicin, and spectinomycin), amphenicol antibiotics(e.g., azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol),ansamycin antibiotics (e.g., rifamide and rifampin), carbacephems (e.g.,loracarbef), carbapenems (e.g., biapenem and imipenem), cephalosporins(e.g., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone,cefozopran, cefpimizole, cefpiramide, and cefpirome), cephamycins (e.g.,cefbuperazone, cefmetazole, and cefminox), monobactams (e.g., aztreonam,carumonam, and tigemonam), oxacephems (e.g., flomoxef, and moxalactam),penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin,bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium,epicillin, fenbenicillin, floxacillin, penamccillin, penethamatehydriodide, penicillin o-benethamine, penicillin 0, penicillin V,penicillin V benzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), macrolides (e.g., azithromycin, carbomycin, clarithomycin,dirithromycin, erythromycin, and erythromycin acistrate), amphomycin,bacitracin, capreomycin, colistin, enduracidin, enviomycin,tetracyclines (e.g., apicycline, chlortetracycline, clomocycline, anddemeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans(e.g., furaltadone, and furazolium chloride), quinolones and analogsthereof (e.g., cinoxacin, ciprofloxacin, clinafloxacin, flumequine, andgrepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine,benzylsulfamide, noprylsulfamide, phthalylsulfacetamide,sulfachrysoidine, and sulfacytine), sulfones (e.g., diathymosulfone,glucosulfone sodium, and solasulfone), cycloserine, mupirocin andtuberin.

Additional examples of antibacterial agents include but are not limitedto Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin;Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin Mesylate;Amikacin; Amikacin Sulfate; Aminosalicylic acid; Aminosalicylate sodium;Amoxicillin; Amphomycin; Ampicillin; Ampicillin Sodium; ApalcillinSodium; Apramycin; Aspartocin; Astromicin Sulfate; Avilamycin;Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium; BacampicillinHydrochloride; Bacitracin; Bacitracin Methylene Disalicylate; BacitracinZinc; Bambermycins; Benzoylpas Calcium; Berythromycin; BetamicinSulfate; Biapenem; Biniramycin; Biphenamine Hydrochloride; BispyrithioneMagsulfex; Butikacin; Butirosin Sulfate; Capreomycin Sulfate; Carbadox;Carbenicillin Disodium; Carbenicillin Indanyl Sodium; CarbenicillinPhenyl Sodium; Carbenicillin Potassium; Carumonam Sodium; Cefaclor;Cefadroxil; Cefamandole; Cefamandole Nafate; Cefamandole Sodium;Cefaparole; Cefatrizine; Cefazaflur Sodium; Cefazolin; Cefazolin Sodium;Cefbuperazone; Cefdinir; Cefepime; Cefepime Hydrochloride; Cefetecol;Cefixime; Cefmnenoxime Hydrochloride; Cefinetazole; Cefinetazole Sodium;Cefonicid Monosodium; Cefonicid Sodium; Cefoperazone Sodium; Ceforanide;Cefotaxime Sodium; Cefotetan; Cefotetan Disodium; CefotiamHydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimizole; CefpimizoleSodium; Cefpiramide; Cefpiramide Sodium; Cefpirome Sulfate; CefpodoximeProxetil; Cefprozil; Cefroxadine; Cefsulodin Sodium; Ceftazidime;Ceftibuten; Ceftizoxime Sodium; Ceftriaxone Sodium; Cefuroxime;Cefuroxime Axetil; Cefuroxime Pivoxetil; Cefuroxime Sodium; CephacetrileSodium; Cephalexin; Cephalexin Hydrochloride; Cephaloglycin;Cephaloridine; Cephalothin Sodium; Cephapirin Sodium; Cephradine;Cetocycline Hydrochloride; Cetophenicol; Chloramphenicol;Chloramphenicol Palmitate; Chloramphenicol Pantothenate Complex;Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate;Chloroxylenol; Chlortetracycline Bisulfate; ChlortetracyclineHydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride;Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; CloxacillinSodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin;Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone;Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline;Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium;Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline;Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; DroxacinSodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride;Erythromycin; Erythromycin Acistrate; Erythromycin Estolate;Erythromycin Ethylsuccinate; Erythromycin Gluceptate; ErythromycinLactobionate; Erythromycin Propionate; Erythromycin Stearate; EthambutolHydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid;Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin;Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole;Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin;Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin;Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride;Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; MeclocyclineSulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem;Methacycline; Methacycline Hydrochloride; Methenamine; MethenamineHippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim;Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin;Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; MirincamycinHydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; NalidixateSodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate;Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate;Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone;Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole;Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium;Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium;Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; OxytetracyclineHydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin;Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin GPotassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V;Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin VPotassium; Pentizidone Sodium; Phenyl Aminosalicylate; PiperacillinSodium; Pirbenicillin Sodium; Piridicillin Sodium; PirlimycinHydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin;Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin;Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin;Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin;Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; RosaramicinButyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate;Rosaramicin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline;Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafingin; Sisomicin;Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride;Spiramycin; Stallimycin Hydrochloride; Steffimycin; StreptomycinSulfate; Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide;Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium;Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine;Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole;Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole;Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl;Sulfisoxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; SuncillinSodium; Talampicillin Hydrochloride; Teicoplanin; TemafloxacinHydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride;Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol;Thiphencillin Potassium; Ticarcillin Cresyl Sodium; TicarcillinDisodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride;Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; TrimethoprimSulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate;Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin;Zorbamycin.

Antibiotic therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physicians' Desk Reference (58th ed., 2004) and MerckVeterinary Manual (8th ed., 1998).

5.7.5 Immunomodulatory Therapies

Any immunomodulatory agent well-known to one of skill in the art may beused in the methods and compositions of the invention. Immunomodulatoryagents can affect one or more or all aspects of the immune response in asubject. Aspects of the immune response include, but are not limited to,the inflammatory response, the complement cascade, leukocyte andlymphocyte differentiation, proliferation, and/or effector function,monocyte and/or basophil counts, and the cellular communication amongcells of the immune system. In certain embodiments of the invention, animmunomodulatory agent modulates one aspect of the immune response. Inother embodiments, an immunomodulatory agent modulates more than oneaspect of the immune response. In a preferred embodiment of theinvention, the administration of an immunomodulatory agent to a subjectinhibits or reduces one or more aspects of the subject's immune responsecapabilities. In certain embodiments, an immunomodulatory agent is notan anti-inflammatory agent. In certain embodiments, an immunomodulatoryagent is not an anti-angiogenic agent. In certain embodiments, animmunomodulatory agent is a chemotherapeutic agent. In certainembodiments, an immunomodulatory agent is not a chemotherapeutic agent.

Examples of immunomodulatory agents include, but are not limited to,proteinaceous agents such as cytokines, peptide mimetics, and antibodies(e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs,Fab or F(ab)2 fragments or epitope binding fragments), nucleic acidmolecules (e.g., antisense nucleic acid molecules and triple helices),small molecules, organic compounds, and inorganic compounds. Inparticular, immunomodulatory agents include, but are not limited to,methotrexate, leflunomide, cyclophosphamide, cytoxan, Immuran,cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506(tacrolimus)), methylprednisolone (MP), corticosteroids, steroids,mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), Tcell receptor modulators, and cytokine receptor modulators.

Examples of T cell receptor modulators include, but are not limited to,anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g.,cM-T412 (Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthocloneand OKTcdr4a (Janssen-Cilag)), anti-CD2 antibodies, anti-CD3 antibodies(e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), orRituxan (IDEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linkedimmunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)),anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g.,IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)),anti-CD2 antibodies, anti-CD11a antibodies (e.g., Xanelim (Genentech)),and anti-B7 antibodies (e.g., IDEC-114) (IDEC))), CTLA4-immunoglobulin,and LFA-3TIP (Biogen, International Publication No. WO 93/08656 and U.S.Pat. No. 6,162,432).

Examples of cytokine receptor modulators include, but are not limitedto, soluble cytokine receptors (e.g., the extracellular domain of aTNF-α receptor or a fragment thereof, the extracellular domain of anIL-1β receptor or a fragment thereof, and the extracellular domain of anIL-6 receptor or a fragment thereof), cytokines or fragments thereof(e.g., interleukin IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-15, IL-23, TNF-α, TNF-β, interferon(IFN)-α, IFN-β, IFN-γ, and GM-CSF), anti-cytokine receptor antibodies(e.g., anti-IFN receptor antibodies, anti-IL-2 receptor antibodies(e.g., Zenapax (Protein Design Labs)), anti-IL-3 receptor antibodies,anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-9receptor antibodies anti-IL-10 receptor antibodies, anti-IL-12 receptorantibodies, anti-IL-13 receptor antibodies, anti-IL-15 receptorantibodies, and anti-IL-23 receptor antibodies), anti-cytokineantibodies (e.g., anti-IFN antibodies, anti-TNF-α antibodies, anti-IL-1βantibodies, anti-IL-3 antibodies, anti-IL-6 antibodies, anti-IL-8antibodies (e.g., ABX-IL-8 (Abgenix)), anti-IL-9 antibodies anti-IL-12antibodies, anti-IL-13 antibodies, anti-IL-15 antibodies, and anti-IL-23antibodies).

In a specific embodiment, a cytokine receptor modulator is IL-3, IL-4,IL-10, or a fragment thereof. In another embodiment, a cytokine receptormodulator is an anti-IL-1β antibody, anti-IL-6 antibody, anti-IL-12receptor antibody, or anti-TNF-α antibody. In another embodiment, acytokine receptor modulator is the extracellular domain of a TNF-αreceptor or a fragment thereof.

In certain embodiments, an immunomodulatory agent is a B cell receptormodulator. Examples of B cell receptor modulators include, but are notlimited to, CD19. Targeting B cell receptors provides one means formodulating B cells.

In a preferred embodiment, proteins, polypeptides or peptides (includingantibodies) that are utilized as immunomodulatory agents are derivedfrom the same species as the recipient of the proteins, polypeptides orpeptides so as to reduce the likelihood of an immune response to thoseproteins, polypeptides or peptides. In another preferred embodiment,when the subject is a pig, the proteins, polypeptides, or peptides thatare utilized as immunomodulatory agents are derived from pig.

In accordance with the invention, one or more immunomodulatory agentsare administered to a subject prior to, subsequent to, or concomitantlywith an attenuated swine influenza virus. Any technique well-known toone skilled in the art can be used to measure one or more aspects of theimmune response in a particular subject, and thereby determine when itis necessary to administer an immunomodulatory agent to said subject. Ina preferred embodiment, a mean absolute lymphocyte count ofapproximately 500 cells/mm³, preferably 600 cells/mm³, 650 cells/mm³,700 cells/mm³, 750 cells/mm³, 800 cells/mm³, 900 cells/mm³, 1000cells/mm³, 1100 cells/mm³, or 1200 cells/mm³ is maintained in a subject.

Preferably, agents that are commercially available and known to functionas immunomodulatory agents are used in accordance with the methods ofthe invention. The immunomodulatory activity of an agent can bedetermined in vitro and/or in vivo by any technique well-known to oneskilled in the art, including, e.g., by CTL assays, proliferationassays, and immunoassays (e.g. ELISAs) for the expression of particularproteins such as co-stimulatory molecules and cytokines.

5.8 Dosage & Frequency of Administration

The amount of a prophylactic or therapeutic agent or a composition ofthe invention which will be effective in the prevention, treatment,management, and/or amelioration of a swine influenza virus infection ora condition associated therewith, an infection other than a swineinfluenza virus infection or a condition associated therewith, anIFN-treatable disease or a condition in which an attenuated swineinfluenza virus of the invention is used as a vector to induce an immuneresponse to an antigen associated with the condition, or the preventionof the recurrence, onset, or development of one or more symptoms of aswine influenza virus infection or a condition associated therewith, aninfection other than a swine influenza virus infection or a conditionassociated therewith, an IFN-treatable disease, or a condition in whichan attenuated swine influenza virus of the invention is used as a vectorto induce an immune response to an antigen associated with thecondition, can be determined by standard clinical methods. The frequencyand dosage will vary also according to factors specific for each patientdepending on the specific therapies (e.g., the specific therapeutic orprophylactic agent or agents) administered, the severity of thedisorder, disease, or condition, the route of administration, as well asage, body, weight, response, and the past medical history of thepatient. For example, the dosage of a prophylactic or therapeutic agentor a composition of the invention which will be effective in thetreatment, prevention, management, and/or amelioration of a swineinfluenza virus infection or a condition associated therewith, a viralinfection other than a swine influenza virus infection or a conditionassociated therewith, or an IFN-treatable disease, or the prevention ofthe recurrence, onset, or development of one or more symptoms of a swineinfluenza virus infection or a condition associated therewith, a viralinfection other than a swine influenza virus infection or a conditionassociated therewith, or an IFN-treatable disease, can be determined byanimal models such as those known to those skilled in the art. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. Suitable regimens can be selected by one skilledin the art by considering such factors and by following, for example,dosages are reported in literature and recommended in the Physician'sDesk Reference (58th ed., 2004), Merck Veterinary Manual (8th ed.,1998), or Straw et al., Diseases of the Swine, Iowa State UniversityPress (1999).

Exemplary doses of a vaccine or immunogenic formulation include 10² toabout 10⁸, about 10³ to about 10⁷, about 10⁴ to about 5×10⁶ pfu or 10⁴to about 10⁷ pfu. In other embodiments, the dose of a vaccine orimmunogenic formulation of the invention administered to a subject is10², 5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷or 10⁸ pfu. Exemplary doses of a pharmaceutical formulation include 10²,5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷, 10⁸,5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or 10¹² pfu. Incertain embodiments, the dose of the pharmaceutical formulationadministered to the subject is between about 10² to about 10¹², about10² to about 10¹⁰, about 10² to about 10⁸, about 10³ to about 10⁹, about10³ to about 10⁷, about 10⁴ to about 10⁸, about 10⁴ to about 5×10⁶ pfuor about 10⁴ to about 10¹² pfu.

Exemplary doses of a small molecule include milligram or microgramamounts of the small molecule per kilogram of subject or sample weight(e.g., about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 5 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram).

For antibodies, proteins, polypeptides, peptides and fusion proteinsencompassed by the invention, the dosage administered to a patient istypically 0.0001 mg/kg to 100 mg/kg of the patient's body weight.Preferably, the dosage administered to a patient is between 0.0001 mg/kgand 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg,0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg,0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or0.01 to 0.10 mg/kg of the patient's body weight. Generally, pigantibodies have a longer half-life within the pig than antibodies fromother species due to the immune response to the foreign polypeptides.Thus, lower dosages of pig antibodies and less frequent administrationto pigs is often possible. Further, the dosage and frequency ofadministration of antibodies or fragments thereof may be reduced byenhancing uptake and tissue penetration of the antibodies bymodifications such as, for example, lipidation.

Preferably, the dosages of prophylactic or therapeutic agents used incombination therapies of the invention are lower than those which havebeen or are currently being used to prevent, treat, manage, and/orameliorate a swine influenza virus infection or a condition or symptomsassociated therewith, an infection other than a swine influenza virusinfection or a condition or symptom associated therewith, an IFNtreatable disease, or a condition in which an attenuated swine influenzavirus of the invention is used as a vector to induce an immune responseto an antigen associated with the condition. The recommended dosages ofagents currently used for the prevention, treatment, management, oramelioration of a swine influenza virus infection or a condition orsymptoms associated therewith, an infection other than a swine influenzavirus infection or a condition or symptom associated therewith, an IFNtreatable disease, or a condition in which an attenuated swine influenzavirus of the invention is used as a vector to induce an immune responseto an antigen associated with the condition, can be obtained from anyreference in the art including, but not limited to, Hardman et al.,eds., 2001, Goodman & Gilman's The Pharmacological Basis Of Basis OfTherapeutics, 10th ed., Mc-Graw-Hill, New York; Physicians' DeskReference (PDR) 58th ed., 2004, Medical Economics Co., Inc., Montvale,N.J., and Merck Veterinary Manual (8th ed., 1998); Taylor, Pig Diseases,6^(th) Ed., Diamond Farm Book Pubns, 1995; and Straw et al., Diseases ofSwine, 8^(th) Ed., Iowa State University Press, 1999, which areincorporated by reference herein in their entireties.

In various embodiments, the therapies (e.g., prophylactic or therapeuticagents) are administered less than 5 minutes apart, less than 30 minutesapart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hoursapart, at about 2 hours to about 3 hours apart, at about 3 hours toabout 4 hours apart, at about 4 hours to about 5 hours apart, at about 5hours to about 6 hours apart, at about 6 hours to about 7 hours apart,at about 7 hours to about 8 hours apart, at about 8 hours to about 9hours apart, at about 9 hours to about 10 hours apart, at about 10 hoursto about 11 hours apart, at about 11 hours to about 12 hours apart, atabout 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hoursto 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hoursapart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hoursto 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hourspart. In preferred embodiments, two or more therapies are administeredwithin the same patent visit.

In certain embodiments, one or more compositions of the invention andone or more other therapies (e.g., prophylactic or therapeutic agents)are cyclically administered. Cycling therapy involves the administrationof a first therapy (e.g., a first prophylactic or therapeutic agent) fora period of time, followed by the administration of a second therapy(e.g., a second prophylactic or therapeutic agent) for a period of time,optionally, followed by the administration of a third therapy (e.g.,prophylactic or therapeutic agent) for a period of time and so forth,and repeating this sequential administration, i.e., the cycle in orderto reduce the development of resistance to one of the therapies, toavoid or reduce the side effects of one of the therapies, and/or toimprove the efficacy of the therapies.

In a certain other embodiments, a vaccine or immunogenic composition ofthe invention is administered to a subject at a single dose followed bya second dose 3 to 6 weeks later. In accordance with these embodiments,booster vaccinations are administered to the subject at 6 to 12 monthintervals following the second vaccination. In a preferred embodiment,the subject is a mammal. In a more preferred embodiment, the subject isa pig with an swine influenza A infection.

In certain embodiments, the administration of the same compostions ofthe invention may be repeated and the administrations may be separatedby at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45days, 2 months, 75 days, 3 months, or at least 6 months. In otherembodiments, the administration of the same therapy (e.g., prophylacticor therapeutic agent) other than a composition of the invention may berepeated and the administration may be separated by at least at least 1day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2months, 75 days, 3 months, or at least 6 months.

5.9 Biological Assays

5.9.1 In Vitro Assays

Growth of the attenuated swine influenza viruses of the presentinvention can be assessed in pig cells, e.g., PK cells, other pig cellsas described herein, and other IFN-competent and IFN-deficient cells. Ina specific embodiment, PK cells are infected at a MOI of 0.0005 and0.001, 0.001 and 0.01, 0.01 and 0.1, 0.1 and 1, or 1 and 10, or a MOI of0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 or 10 and incubatedwith serum free media supplemented with 5% allantoic fluid (F. Cook,University of Kentucky, Lexington, Ky.). Viral titers are determined inthe supernatant by HA plagued in MDCK cells as described below. Othercells in which viral titers can be assessed include, but are not limitedto, PK cells, Vero cells, primary human umbilical vein endothelial cells(HUVEC), H292 human epithelial cell line, HeLa cells, and swineembryonic kidney cells RES.

Viral assays include those that measure altered viral replication (asdetermined, e.g., by plaque formation) or the production of viralproteins (as determined, e.g., by western blot analysis) or viral RNAs(as determined, e.g., by RT-PCR or northern blot analysis) in culturedcells in vitro using methods which are well known in the art.

Induction of IFN responses may be determined by measuring thephosphorylated state of components of the IFN pathway followinginfection with the test mutant virus, e.g., IRF-3, which isphosphorylated in response to double-stranded RNA. In response to type IIFN, Jak1 kinase and TyK2 kinase, subunits of the IFN receptor, STAT1,and STAT2 are rapidly tyrosine phosphorylated. Thus, in order todetermine whether the attenuated swine influenza virus induces IFNresponses, cells, such as 293 cells, are infected with the test mutantvirus and following infection, the cells are lysed. IFN pathwaycomponents, such as Jak1 kinase or TyK2 kinase, are immunoprecipitatedfrom the infected cell lysates, using specific polyclonal sera orantibodies, and the tyrosine phosphorylated state of the kinase isdetermined by immunoblot assays with an anti-phosphotyrosine antibody(e.g., see Krishnan et al. 1997, Eur. J. Biochem. 247: 298-305). Anenhanced phosphorylated state of any of the components of the IFNpathway following infection with the attenuated swine influenza viruswould indicate induction of IFN responses by the attenuated swineinfluenza virus.

Induction of IFN responses may be determined by measuring IFN-dependenttranscriptional activation following infection with the test attenuatedswine influenza virus. In this embodiment, the expression of genes knownto be induced by IFN, e.g., Mx, PKR, 2-5-oligoadenylatesynthetase, majorhistocompatibility complex (MHC) class I, etc., can be analyzed bytechniques known to those of skill in the art (e.g., northern blots,western blots, PCR, etc.). Alternatively, test cells such as embryonickidney cells or osteogenic sarcoma cells, are engineered to transientlyor constitutively express reporter genes such as luciferase reportergene or chloramphenicol transferase (CAT) reporter gene under thecontrol of an interferon stimulated response element, such as theIFN-stimulated promoter of the ISG-54K gene (Bluyssen et al., 1994, Eur.J. Biochem. 220:395-402). Cells are infected with the test attenuatedswine influenza virus and the level of expression of the reporter genecompared to that in uninfected cells or cells infected with wild-typevirus. An increase in the level of expression of the reporter genefollowing infection with the test virus would indicate that the testattenuated swine influenza virus is inducing an IFN response.

Measuring IFN induction can also be assessed by determining whether anextract from the cell or egg infected with the test attenuated swineinfluenza virus is capable of conferring protective activity againstviral infection. More specifically, groups of 10-12 day old embryonatedchicken eggs or 10-day old embryonated chicken eggs are infected withthe test attenuated swine influenza virus or the wild-type virus.Approximately 15 to 20 hours post infection, the allantoic fluid isharvested and tested for IFN activity by determining the highestdilution with protective activity against swine influenza virusinfection in tissue culture cells, such as MDCK cells.

5.9.2 In Vivo Assays

The decreased virulence of the attenuated swine influenza viruses of thepresent invention can be assessed in a subject, in particular, pigs. Inone example, the ability to induce lung lesions and cause infection inswine is compared to wild-type swine influenza virus and mock virus.Lung lesions can be assessed as a percentage of lung lobes that arehealthy by visual inspection. Animals are euthanized 5 days p.i. byintravenous administration of pentobarbital, and their lungs are removedin toto. The percentage of the surface of each pulmonary lobe that isaffected by macroscopic lesions is estimated visually. The percentagesare averaged to obtain a mean value for the 7 pulmonary lobes of eachanimal.

In other assays, nasal swabs and BALF can be tested to determine virusburden or titer. Nasal swabs can be taken during necropsy to determineviral burden post-infection. Samples can also be taken with care fromlive pigs to determine viral burden post-infection. BALF can be obtainedafter the lungs are removed from the thoracic cavity by rinsing thelungs (via trachea) with about 30 ml of McCoy's medium without serum.

For quantitation of virus in tissue samples, tissue samples arehomogenized in phosphate-buffered saline (PBS), and dilutions ofclarified homogenates adsorbed for 1 h at 37° C. onto monolayers of MDCKcells. Infected monolayers are then overlaid with a solution of minimalessential medium containing 0.1% bovine serum albumin (BSA), 0.01%DEAE-dextran, 0.1% NaHCO₃, and 1% agar. Plates are incubated 2 to 3 daysuntil plaques could be visualized. Tissue culture infectious dose (TCID)assays to titrate virus from PR8-infected samples are carried out asfollows. Confluent monolayers of MDCK cells in 96-well plates areincubated with log dilutions of clarified tissue homogenates in media.Two to three days after inoculation, 0.05-ml aliquots from each well areassessed for viral growth by hemagglutination assay (HA assay).

In yet other assays, histopathologic evaluations are performed afterinfection. The nasal turbinates and trachea are examined for epithelialchanges and subepithelial inflammation. The lungs are examined forbronchiolar epithelial changes and peribronchiolar inflammation inlarge, medium, and small or terminal bronchioles. The alveoli are alsoevaluated for inflammatory changes. The medium bronchioles are graded ona scale of 0 to 3+ as follows: 0 (normal: lined by medium to tallcolumnar epithelial cells with ciliated apical borders and basalpseudostratifled nuclei; minimal inflammation); 1+ (epithelial layercolumnar and even in outline with only slightly increased proliferation;cilia still visible on many cells); 2+ (prominent changes in theepithelial layer ranging from attenuation to marked proliferation; cellsdisorganized and layer outline irregular at the luminal border); 3+(epithelial layer markedly disrupted and disorganized with necroticcells visible in the lumen; some bronchioles attenuated and others inmarked reactive proliferation).

The trachea is graded on a scale of 0 to 2.5+ as follows: 0 (normal:Lined by medium to tall columnar epithelial cells with ciliated apicalborder, nuclei basal and pseudostratified. Cytoplasm evident betweenapical border and nucleus. Occasional small focus with squamous cells);1+ (focal squamous metaplasia of the epithelial layer); 2+ (diffusesquamous metaplasia of much of the epithelial layer, cilia may beevident focally); 2.5+ (diffuse squamous metaplasia with very few ciliaevident).

Swine influenza virus immunohistochemistry is performed using aNP-specific monoclonal antibody. Staining is graded 0 to 3+ as follows:0 (no infected cells); 0.5+ (few infected cells); 1+ (few infectedcells, as widely separated individual cells); 1.5+ (few infected cells,as widely separated singles and in small clusters); 2+ (moderate numbersof infected cells, usually affecting clusters of adjacent cells inportions of the epithelial layer lining bronchioles, or in smallsublobular foci in alveoli); 3+ (numerous infected cells, affecting mostof the epithelial layer in bronchioles, or widespread in largesublobular foci in alveoli).

5.9.3 Determining Viral Titer

Viral titer is determined by inoculating serial dilutions of swineinfluenza virus into cell cultures (e.g., pig cultures), chick embryos,or live animals (e.g., pigs). After incubation of the virus for aspecified time, the virus is isolated using standard methods.

Physical quantitation of the virus titer can be performed using PCRapplied to viral supernatants (Quinn & Trevor, 1997; Morgan et al.,1990), hemagglutination assays, tissue culture infectious doses (TCID50)or egg infectious doses (EID50).

The HA assay is carried out in V-bottom 96-well plates. Serial twofolddilutions of each sample in PBS were incubated for 1 h on ice with anequal volume of a 0.5% suspension of chicken erythrocytes in PBS.Positive wells contained an adherent, homogeneous layer of erythrocytes;negative wells contain a nonadherent pellet.

5.9.4 Determining Hemagglutination Inhibiting Antibody Titers

In one method, HA inhibition assay, the levels of hemagglutination(HA)-inhibiting (HI) antibodies in samples are determined as describedpreviously (Palmer et al., 1975, Advanced laboratory techniques forinfluenza diagnosis. U.S. Department of Health, Education and WelfareImmunology Series. U.S. Department of Health, Education and Welfare,Washington, D.C.). Briefly, samples are incubated overnight at 37° C.with 4 volumes of receptor-destroying enzyme (Denka Seiken Co., Tokyo,Japan) prepared from Vibrio cholerae. After inactivation of thereceptor-destroying enzyme by incubation of the samples at 56° C. for 60min, twofold serial dilutions of sera are mixed with 4 HA units of swineinfluenza virus. The assays are developed by adding 0.5% (vol/vol)chicken red blood cells, and the HI antibody titers are defined as thereciprocal of the highest dilution causing complete inhibition ofagglutination.

5.9.5 Toxicity Studies

The toxicity and/or efficacy of the compositions of the presentinvention can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Therapies that exhibit largetherapeutic indices are preferred. While therapies that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such agents to the site of affected tissue in orderto minimize potential damage to uninfected cells and, thereby, reduceside effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the therapies for use insubjects (e.g., pigs). The dosage of such agents lies preferably withina range of circulating concentrations that include the ED50 with littleor no toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anytherapy used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in subjects (e.g., pigs). Levels inplasma may be measured, for example, by high performance liquidchromatography.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of a composition, acombination therapy disclosed herein for swine influenza virus infectionor a condition or symptoms associated therewith, an infection other thana swine influenza virus infection or a condition or symptom associatedtherewith, an IFN treatable disease, or a condition in which anattenuated swine influenza virus of the invention is used as a vector toinduce an immune response to an antigen associated with the condition.

The following examples are provided to further illustrate the currentinvention but are not provided to in any way limit the scope of thecurrent invention.

6. EXAMPLES 6.1 Example 1 Generation of Plasmid-Derived Sw/Tx98 VirusesEncoding Truncated NS1

Site directed mutagenesis was used to generate different deletions inthe swine influenza virus NS segment in such a way that the NEP wasexpressed without any alteration while the NS1 was partially deleted.This was achieved by inserting a stop codon in the NS1 ORF followed by adeletion in the NS1 ORF encompassing nucleotides not involved in NEPexpression.

Cells and Viruses.

Pig kidney-15 (PK-15), Swine Testis (ST) and Madin-Darby canine kidney(MDCK) type II cells were grown in minimal essential medium (MEM)supplemented with 10% fetal bovine serum (FBS). Baby hamster kidney(BHK), 293T and A549 cells were grown in DMEM with 10% FBS. All cellswere maintained at 37° C. and 5% CO₂. The A/Swine/Texas/4199-2/98(TX/98, H3N2 subtype) influenza virus was obtained from the repositoryat St. Jude Children's Research Hospital, Memphis, Tenn. Influenzaviruses were grown in the allantoic cavities of embryonated chicken eggsor in MDCK cells. Recombinant vesicular stomatitis virus expressing GFP(VSV-GFP) (Stojdl, 2003, Cancer Cell 4:263-75) was grown and titrated inBHK cells for use in IFN bioassays.

Construction of Plasmids.

MDCK cells were infected with TX/98 virus and total RNA was extracted byusing Trizol reagent according to the manufacturer's instructions(Invitrogen). The eight reverse genetics plasmids pHW-Sw-PB2,pHW-Sw-PB1, pHW-Sw-PA, pHW-Sw-HA, pHW-Sw-NP, pHW-Sw-NA, pHW-Sw-M andpHW-Sw-NS (corresponding to the eight influenza viral segments listed inTable 1) were constructed by RT-PCR amplification of single viral RNAsegments and cloning of the resulting cDNAs into the vector pHW2000(Hoffmann et al., 2000, Proc. Natl. Acad. Sci. USA 97:6108-6113). Theinsert sequences for pHW-Sw-PB2, pHW-Sw-PB1, pHW-Sw-PA, pHW-Sw-HA,pHW-Sw-NP, pHW-Sw-NA, pHW-Sw-M and pHW-Sw-NS are provided in SEQ ID NOS:1-8, respectively. In this system, influenza viral cDNA is insertedbetween the RNA polymerase I (polI) promoter and terminator sequences.This entire RNA polI transcription unit is flanked by an RNA polymeraseII (polII) promoter and a polyadenylation site. The orientation of thetwo promoters allows the synthesis of negative-sense viral RNA andpositive-sense mRNA from one viral cDNA template. pHW-Sw-NS-73,pHW-Sw-NS-99 and pHW-Sw-NS-126 are derivatives of pHW-Sw-NS. These NS1mutant plasmids were constructed by trimolecular ligation: Two PCRproducts were ligated into SalI/NgoIV digested pHW2000-Sw-NS.

The first PCR product was common for all three NS1 mutant plasmids, andwas obtained using oligo pHW-3′ (reverse, annealing in the plasmidpHW2000 backbone) GGGTCAAGGAAGGCACGGGGGAGGGGC (SEQ ID NO: 9) and oligo5NS-PacI (forward, annealing in the NS1 gene)GCGCTTAATTAAGAGGGAGCAATCGTTGGAG (SEQ ID NO: 10) and pHW2000-Sw-NS astemplate. This PCR product was digested with NgoIV and PacI.

The second PCR product was specific for each NS1 mutant plasmid, and wasobtained using: a common forward primer, CMV5′ (annealing in the CMVpromoter of pHW2000 plasmid) GCCCCATTGACGCAAATGGGCGGTAGGCGTG (SEQ ID NO:11), and a specific reverse primer (annealing in the NS1 gene):NS73-BglII-PacI-3′ GCGCTTAATTAATCAAGATCTAGGATTCCTCTTTCAAAATCC (SEQ IDNO: 12), NS99-BglII-PacI-3′ GCTTAATTAATCAAGATCTATGACATTTCCTCGAGGGTCATG(SEQ ID NO: 13) or NS126-BglII-PacI-3′GCGCTTAATTAATCAAGATCTACTTTTCCATGATCGCCTGGTCC (SEQ ID NO: 14). The threecorresponding PCR products were digested with SalI and PacI, and used intrimolecular ligation together with the PacI/NgoIV digested first PCRproduct and SalI/NgoIV digested pHW2000-Sw-NS, to generate:pHW-Sw-Tx-73, pHW-Sw-Tx-99 and pHW-Sw-Tx-126. These constructs contain adeletion in the NS1 sequence, plus the insertion of 4 stop codons in the3 frames after this deletion. See FIG. 1A. While the nuclear exportprotein (NEP) ORF is not altered in these constructs, the NS1 ORFencodes only the first 73, 99 and 126 amino acids of the wild-type NS1protein (total length of wild type NS1 is 219 amino acids),respectively.

The viral strains are described below:

Sw/TX/98 wt: contains wild-type NS1 gene and represents atriple-reassortant H3N2 swine influenza virus.

Sw/TX/98/del126: expresses the N-terminal 126 amino acids of thewild-type NS1 protein.

Sw/TX/98/del99: expresses the N-terminal 99 amino acids of the wild-typeNS1 protein.

Sw/TX/98/del73: expresses the N-terminal 72 amino acids of the wild-typeNS1 protein.

Transfection-Mediated Recovery of Recombinant SIV.

Rescue of influenza viruses from plasmid DNA was performed as describedin Fodor et al. (1999, J Virol 73:9679-82) and Neumann et al. (1999,Proc Natl Acad Sci USA 96:9345-50) using an eight-plasmid system(Hoffmann et al., 2000, Proc. Natl. Acad. Sci. USA 97:6108-6113). Togenerate the recombinant wild type (rWT) TX/98 virus 0.5 μg of each ofthe 8 pHW plasmids were transfected into 10⁶ 293 T cells in suspensionusing the Lipofectamine 2000 reagent (Invitrogen). The NS1 truncatedmutant viruses were generated in the same way but substituting thepHW-Sw-NS plasmid by the corresponding mutant one to recover the del73,del99 or del126 virus mutants. The resulting viruses were passaged andcloned by plaque purification into MDCK cells. Virus stocks were grownin 7-day-old embryonated chicken eggs. The identity of the recombinantviruses SIV was verified by gel electrophoretic analyses of RT-PCRproducts derived from viral RNA using oligonucleotides specific for thecommon non-coding regions. The wild type NS gene or the deleted versionswere further confirmed by sequencing.

Results.

To generate SIV using reverse genetics, the eight viral RNAs from Tx/98virus were cloned into ambisense expression plasmids for viral rescue(Hoffmann et al., 2000, Proc. Natl. Acad. Sci. USA 97:6108-6113). Upontransfection of these plasmids in 293T cells, infectious viruses wererecovered. The parental (WT) and the plasmid-derived wild type (rWT)SIVs grew to similar titers in MDCK cells and embryonated eggs. Wheninoculated into pigs, the rWT virus produced similar lesions as theparental WT virus. Three NS1-truncated SIV mutants were generatedencoding NS1 proteins of 73, 99 and 126 amino acids, as compared to thefull length, 219 amino acids long, NS1 protein (FIG. 1A). RT-PCR andsequencing analyses confirmed the presence of the truncated NS1 genes inthe rescued virus preparations (FIG. 1B).

6.2 Example 2 Characterization of the Swine Influenza Virus NS1 DeletionMutants and Wild Type A/Swine/Texas/4199-2/98

To characterize the impact of the NS1 gene deletion on the replicativeproperties of swine influenza virus, the growth of the wild-type and thedifferent deletion mutants described in Section 6.1 was compared. Also,the effect of these mutants on IFN production was compared.

Virus Growth Curves.

To analyze viral replication, confluent PK-15 cells were infected at theindicated multiplicity of infection (MOI) and incubated for differentperiods of times at 37° C. in MEM containing 0.3% bovine albumin(MEM/BA) and 5% allantoic fluid. Virus titers were determined by plaqueassay on MDCK cells in MEM/BA supplemented with 1 μg/ml of TPCK trypsin.Titers were expressed as plaque forming units (PFU) per ml.

In order to investigate the multicycle growth properties of the mutantNS1 viruses confluent pig epithelial cells (PK-15) were infected at alow MOI (MOI=0.001). Supernatants from infected cells were titrated atdifferent time points post infection by plaque assay on MDCK cells. Thegrowth kinetics of the NS1 deletion mutants in PK-15 cells were clearlydifferent when compared to the wild type virus. Interestingly, the 1-126mutant virus was the most compromised in growth, followed by the 1-99and the 1-73 mutants (FIG. 2A). Plaque sizes in MDCK cells correlatedwith the growth differences in PK-15 cells (FIG. 2C). These resultsindicate that deletions in the NS1 protein of Sw/TX/98 virus result inattenuation of viral growth, both in PK-15 and MDCK cells. In contrast,when NS1 mutant viruses were grown at a high MOI (MOI=2) in PK-15 cellsmajor differences in growth kinetics were not detected (FIG. 2B). Thismost likely indicates that virus replication of the NS1 mutant virusesis not restricted during the first cycle of replication, suggesting thatinfected cells secrete antiviral cytokines, such as IFN, that inhibitsubsequent rounds of replication.

Metabolic Labeling.

Confluent PK-15 cells seeded in 22-mm dishes were either mock-infectedor infected with rWT, 1-73, 1-99 or 1-126 viruses at an MOI of 2. Cellswere incubated in MEM/BA at 37° C. for various time points, andsubsequently labeled for 2 h with 10 μCi [³⁵S]Met-Cys in MEM lackingMet-Cys. Cells were washed with ice-cold phosphate-buffered saline(PBS), lysed and total cell extracts were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins werevisualized by autoradiography.

Metabolic labeling of virus-infected PK cells (MOI=2) with[³⁵S]-Met-Cys, revealed no differences in the kinetics of NP proteinsynthesis among rWT and 1-73, 1-99 and 1-126 mutant viruses (FIG. 2D).The NS1 mutant proteins could not be detected by labeling.

Bioassay to Measure IFN Production.

Levels of IFN secreted by virus infected cells were determined aspreviously described (Park et al., 2003, J. Virol. 70:9522-9532;Donelan, 2003, J Virol 77:13257-66), with some variations. ConfluentPK-15 cells seeded in 22-mm dishes were either mock-treated or infectedwith rWT, del73, del99 or del126 viruses at an MOI of 2. Followinginfection, cells were incubated with MEM/BA containing 5% allantoicfluid, and at different time points post-infection, supernatants wereharvested. Viruses present in the supernatant were UV inactivated byplacing samples on ice 6 inches below an 8-W UV lamp (Fisher) for 15 minwith constant stirring. New PK-15 cells were seeded in 96-well platesthe day before and incubated with the UV-inactivated supernatants for 24h. The preincubated PK-15 cells were then infected with VSV-GFP(MOI=0.1). The cells expressing GFP were visualized by fluorescencemicroscopy 16 hours post infection.

In order to see whether the observed attenuation of growth in vitro ofthe NS1-truncated viruses was directly correlated with the ability ofthese viruses to inhibit the IFN-α/β system, the induction of IFN incells infected with the recombinant Sw/Tx/98 viruses was investigated.Supernatants of infected PK-15 cells were used to determine the levelsof secreted IFN-α/β using a bioassay based on inhibition of VSV-GFPreplication (FIG. 3A). For this purpose, PK-15 cells were infected(MOI=2) with rWT and NS1 mutant SIVs and supernatants were collected forIFN determinations every two hours for 10 h. The results are shown inFIG. 3B. Supernatants from mock-infected cells caused no inhibition ofGFP expression by VSV-GFP in PK-15 cells. In contrast, VSV-GFPreplication was completely abolished in cells pretreated with thesupernatant of del126 virus-infected cells at 10 h post infection. NoIFN-α/β was detected by this assay in supernatants of cells infectedwith rWT virus. All NS1-truncated viruses induced detectable levels ofIFN-α/β by 6 h post infection, with 1-126 virus being the strongerIFN-α/β inducer, followed by del99 and del73 viruses. Similarexperiments were performed using other virus-infected swine (ST) andhuman (A549) cell lines with basically identical results.

Analysis of IFN-β and TNF-α mRNA by RT-PCR.

PK-15 cells were infected at an MOI of 2, and at 24 hours postinfection, total RNA was extracted using Absolutely RNA RT-PCR MiniprepKit (Stratagene). RT was performed by using oligodT as the primer. PCRwas done using specific pairs of primers for swine IFN-β (5-SWIFNB+GGCCATGGCTAACAAGTGCATCC (SEQ ID NO: 15), 3-SWIFNB−CCGGTCAGTTCCGGAGGTAATC (SEQ ID NO: 16)) and TNF-α (5′SW-TNFAATGAGCACTGAGAGCATG (SEQ ID NO: 17), 3′SW-TNFA TCACAGGGCAATGATCCC (SEQ IDNO: 18)) mRNAs (Genbank accession numbers M86762 and X57321). Ascontrol, specific primers for β-actin (Donelan, 2003 J Virol77:13257-66) were used to amplify a 550-bp fragment of swine β-actin.The products were sequenced and confirmed to be derived from theexpected mRNAs.

RT-PCR analysis of IFN-β- and TNF-α-transcripts revealed the inductionof the expression of the mRNA of these cytokines in PK-15 cells infectedwith NS1 mutant viruses, especially in 1-126 virus-infected cells (FIG.3C). These data indicate that the potency of inhibition of IFNproduction by the recombinant Sw/Tx/98 viruses is as follows:rWT>del73>del99>del126.

6.3 Example 3 Pathogenesis Of Plasmid-Derived TX/98 Mutant/DeletionViruses

Pathogenesis of the specific swine influenza viruses described inSection 6.1 were assessed in pigs. The ability of Sw/Tx/98/del126,Sw/Tx/98/del99, and Sw/Tx/98/del73 to induce lung lesions and causeinfections in swine were determined and compared to the results obtainedfor a mock virus and the wild-type virus Sw/Tx/98 wt. Lung lesions wereassessed as the percentage of the healthy lung in which lesions occurredwith an average of seven lobes of the lung being used for thisdetermination. Viruses or the mock were administered as described indetail below and either nasal swabs or BALF were obtained duringnecropsy to determine virus burden five days post infection.Histopathologic evaluation was preformed. The nasal turbinates andtrachea were examined for epithelial changes and subepithelialinflammation. The alveoli were also evaluated for inflammatory changes.

This example demonstrates that C-terminal deletions of the NS1 generesult in an attenuated phenotype.

6.3.1 Methods

Infection of Pigs.

Out-bred specific pathogen-free pigs were obtained from a commercial hogfarm in Iowa (USA). Sera from these pigs was confirmed to be negativefor the presence of hemagglutination inhibition antibodies (Palmer,1975, (U.S. Department of Health, Education, and Welfare, Washington,D.C.), pp. 51-52) against H3N2 (Sw/TX/98 and Sw/CO/99) and H1N1(Sw/IA/30) SIV. Groups of four to five pigs were housed in separateisolation rooms. At the age of 4 weeks, pigs were anesthetized byintramuscular injection with a mixture of ketamine, xylazine, zolazepamand tiletamine (Mengeling, 1996, Am J Vet Res 57:834-839) before beinginoculated intratracheally (via laryngoscope and tracheal tube) with 1ml of virus solution containing 1×10⁵ PFU. For 5 days, pigs weremonitored for clinical signs including lethargy, anorexia, coughing,hyperpnea/dyspnea, nasal and ocular discharge and their body temperaturewas recorded daily. At day 5, animals were euthanized by intravenousadministration of pentobarbital (Fort Dodge Animal Health, Fort Dodge,USA), and their lungs were removed in toto. The percentage of thesurface of each pulmonary lobe that was affected by macroscopic lesionswas estimated visually. The percentages were averaged to obtain a meanvalue for the 7 pulmonary lobes of each animal. The nasal turbinates,trachea, and right cardiac lobe were removed and formalin-fixed forfurther histopathologic evaluation. Blood, bronchoalveolar fluid (BALF)and nasal swabs were also collected during necropsy. BALF was obtainedafter the lungs were removed from the thoracic cavity by rinsing thelungs (via trachea) with ca. 30 ml of McCoy's medium without serum. Allanimal experiments were in compliance with the Institutional Animal Careand Use Committee of the National Animal Disease Center.

Clinical Observation and Sampling.

For 5 days, pigs were monitored for clinical signs including lethargy,anorexia, coughing, hyperpnea/dyspnea, nasal and ocular discharge andtheir body temperature was recorded daily. Blood, BALF, and nasal swabswere collected during necropsy. BALF was obtained after the lungs wereremoved from the thoracic cavity by rinsing the lungs (via trachea) withca. 30 ml of McCoy's medium without serum. Nasal swabs and BALF weretested to determine virus burden.

Virus Titrations from Nasal Swabs and BALF.

Nasal swabs and BALF were tested to determine virus burden. Ten-foldserial dilutions were prepared in McCoy's medium without serum,supplemented with 5 μg/ml trypsin. MDCK cells were inoculated with thedilutions and incubated with medium plus trypsin in microtiter plates at37° C. for 72 h. The plates were examined for cytopathic effects after72 hours. Virus titers were calculated by the Reed and Muench method(Reed, 1938, Am J Hyg 27:493-497).

Histopathological Evaluation:

The nasal turbinates and trachea were stained with hematoxylin and eosinand examined under the microscope for epithelial changes andsubepithelial inflammation. The lungs were examined for bronchiolarepithelial changes and peribronchiolar inflammation in large, medium,and small or terminal bronchioles. The alveoli were also evaluated forinflammatory changes. Because lesions were found most consistently inmedium-sized airways, data obtained from the medium bronchioles wereused for comparisons. The medium bronchioles were graded on a scale of 0to 3+ as follows: 0 (normal: lined by medium to tall columnar epithelialcells with ciliated apical borders and basal pseudostratified nuclei;minimal inflammation); 1+ (epithelial layer columnar and even in outlinewith only slightly increased proliferation; cilia still visible on manycells); 2+ (prominent changes in the epithelial layer ranging fromattenuation to marked proliferation; cells disorganized and layeroutline irregular at the luminal border); 3+ (epithelial layer markedlydisrupted and disorganized with necrotic cells visible in the lumen;some bronchioles attenuated and others in marked reactiveproliferation).

The trachea was graded on a scale of 0 to 2.5+ as follows: 0 (normal:Lined by medium to tall columnar epithelial cells with ciliated apicalborder, nuclei basal and pseudostratified. Cytoplasm evident betweenapical border and nucleus. Occasional small focus with squamous cells);1+: (focal squamous metaplasia of the epithelial layer); 2+ (diffusesquamous metaplasia of much of the epithelial layer, cilia may beevident focally); 2.5+ (diffuse squamous metaplasia with very few ciliaevident).

SIV immunohistochemistry was performed using mouse NP-specificmonoclonal antibody HB65 (American Type Culture Collection, Manassas,Va.). The antibody was produced as mouse ascites fluid. A dilution of1/1000 of HB65 was used for immunohistochemistry. The assay wasperformed using the Dako Envision IHC system. Staining was graded 0 to3+ as follows: 0 (no infected cells); 0.5+ (few infected cells); 1+ (fewinfected cells, as widely separated individual cells); 1.5+ (fewinfected cells, as widely separated singles and in small clusters); 2+(moderate numbers of infected cells, usually affecting clusters ofadjacent cells in portions of the epithelial layer lining bronchioles,or in small sublobular foci in alveoli); 3+ (numerous infected cells,affecting most of the epithelial layer in bronchioles, or widespread inlarge sublobular foci in alveoli).

Statistical Analysis.

Mean body temperatures, extent of gross and histopathologic changes, andvirus replication in infected and control groups were compared by usingthe two-sided Student's t-test. Probability (P) values<0.05 wereconsidered to indicate a statistically significant difference betweengroups.

6.3.2 Results

Forty seven 4-week-old outbred pigs were purchased from a commercial hogfarm in Iowa. Groups of 10 pigs were intratracheally infected with 10⁵pfu of rWT and NS1 mutant Sw/TX/98 viruses. Seven animals weremock-infected with medium only. The mean rectal temperature of eachinfected group increased to 40° C. 1 to 3 days p.i. in the rWT, 1-73 and1-99 virus-infected cohorts (data not shown). Not all of the animalsinfected with NS1 deletion mutant 1-126 exhibited increased temperature.Clinical signs, which comprised respiratory distress, nasal secretion,conjunctivitis, and coughing began on days 2 to 4 p.i. in the rWT-virusinfected group. The prevalence of clinical signs differed between therespective viruses. Most clinical signs were consistently observed inthe groups infected with the rWT virus while some signs were observed inthe 1-99 and 1-73 virus-infected cohorts. Only elevated temperature butno other clinical signs were observed in the 1-126 virus-infected pigs.

During necropsy at day 5 p.i., the percentage of each lung surface withmacroscopic lesions was estimated. As shown in FIG. 4A, mock-infectedcontrol animals had no macroscopic lung lesions. Pigs infected with therWT Sw/Tx/98 virus showed significantly higher percentage of macroscopiclung lesions than pigs infected with the NS1 deletion mutant viruses1-99 (p=0.006) or 1-126 (p=0.004). Pigs infected with the NS1 deletionmutant virus 1-73 displayed less severe lesions, however, this was notstatistically not significant (p>0.05). In general, the gross lesions weobserved were marked, plum-colored, consolidated areas on individuallobes. The diaphragmatic lobes were less involved than the other lobes.The mediastinal lymph nodes were usually hyperemic and enlarged.

The medium bronchioles were microscopically examined in tissue sectionsof the right cardiac pulmonary lobe of 4-week-old infected pigs and werehistopathologically scored (FIG. 4B). Mock-infected animals as well as1-126 virus-infected pigs, showed no or minimal lesions, whereasmoderate to severe lesions were detected in animals infected with therWT and the 1-99 and 1-73 Sw/TX/98 viruses (FIGS. 5A-D and 6A-D). Allanimals infected with the parental wild-type and some infected with the1-73 and 1-99 viruses had high scores reflecting disruption of thebronchial epithelial cell layer (characterized by acute epithelialnecrosis or subsequent attenuation or reactive proliferation). Mostanimals infected with either 1-73 or 1-99 virus had a moderate scorereflecting slightly increased proliferation of the bronchial epitheliallayer. The lungs of animals infected with the NS1 deletion mutant 1-126virus were nearly devoid of lesions. As in the macroscopic lung lesions,1-126 and 1-99 infected pigs showed significantly less damage thanrWT-infected pigs (1-126: p<0.0001; 1-99: p=0.0002). Interestingly,microscopic lesions in bronchioles of pigs infected with the 1-73 mutantvirus were also significantly less when compared to the parental rWTvirus (p=0.02).

Virus titers in the BALF were also analyzed at day 5 p.i. As shown inFIG. 4C, all viruses replicated in the respiratory tract of pigs. BALFvirus titers were significantly higher in animals infected with parentalrWT virus when compared to the NS1 deletion mutants. This findingcorrelates with significantly less extensive gross and microscopicdisease observed in pigs infected with NS1 deletion mutants versuswild-type Sw/Tx/98 viruses.

6.4 Example 4 Use of TX/98/del 126 as a Vaccine

The efficacy of the attenuated TX/98/del 126 deletion mutant was testedin a pig vaccination study. Experiments were performed as described inExample 3, except that vaccination with TX/98/del 126 was performedtwice on days 0 and day 21 with 1×10⁵ PFU per pig by intratrachealinoculation. The vaccination and challenge was done by intratrachealinoculation (using 1 ml of virus solution/or medium as a control).Animals were challenged 9 days or 10 days later with the followingpreparations:

-   -   2×10⁵ PFU per pig with wild-type H3N2 virus        (A/Swine/Texas/4199-2/98-homologous challenge)    -   2×10⁵ PFU per pig with wild-type H1N1 virus (A/Swine/MN/37866/99        classical H1N1-heterologous challenge)    -   Mock challenge with 1 ml medium

For histopathological evaluation, the right cardiac lobe of each lungwas stained with hematoxylin and eosin and examined for bronchiolarepithelial changes and peribronchiolar inflammation in large, medium,and small or terminal bronchioles. Because lesions were found mostconsistently in medium-sized airways, data obtained from the mediumbronchioles were used for comparisons. Lesion severity was scored by thedistribution or extent of lesions within the sections examined asfollows: 0—No airways affected. 1—Only a few isolated airways affected;2—Localized cluster of affected airways, often within one or twolobules. 3—Low number of airways affected but throughout much of thesection. 4—Many airways affected, often severely, all sizes.

One trained examiner was utilized for evaluation of tissue sections. Theexaminer was unaware of which group of animals the tissues were derivedfrom.

Immunohistochemistry was done using a monoclonal antibody specific forthe nucleoprotein of influenza. The antigen score was as follows:

0=No antigen positive cells.

1=Only a few cells with positive staining in an occasional airway.

2=Only a few cells with positive staining in scattered airways which maybe localized.

3=Moderate numbers of cells with positive staining in an occasionalairway.

4=Moderate numbers of cells with positive staining in scattered airwaysand alveoli.

Lung lavage was tested to determine virus load. Ten-fold serialdilutions were prepared in McCoy's medium without serum, supplementedwith 5 μg/ml trypsin. MDCK cells were inoculated with the dilutions andincubated with medium plus trypsin in microtiter plates at 37° C. for 72h. The plates were examined for cytopathic effects after 72 hours. Virustiters were calculated by the Reed and Muench method.

Results.

The following results were obtained 5 days after challenge:

-   -   1. Nonvaccinated, nonchallenged pigs did not have any        histopathological changes and did not harbor influenza virus        antigen in their lung tissues (FIGS. 7 and 8). Infectious virus        was not present or below detection limits (FIG. 9).    -   2. Nonvaccinated pigs challenged with wild type H3N2 TX/98 virus        showed significant histopathological damage and influenza viral        antigen in their lung tissues (FIGS. 7 and 8). Virus titers in        lung lavage was ≧10^(6.25) TCID₅₀/ml.    -   3. Nonvaccinated pigs challenged with wild type H1N1 MN/99 virus        showed significant histopathological damage and influenza viral        antigen in their lung tissues (FIGS. 7 and 8). Virus titers in        lung lavage was ≧10^(6.0) TCID₅₀/ml.    -   4. Vaccinated, mock challenged pigs did not have any        histopathological changes and did not harbor influenza virus        antigen in their lung tissues (FIGS. 7 and 8). Infectious virus        was not present or below detection limits (FIG. 9).    -   5. Vaccinated, H3N2 challenged pigs did not have any        histopathological changes and did not harbor influenza virus        antigen in their lung tissues (FIGS. 7 and 8). Infectious virus        was not present or below detection limits (FIG. 9).    -   6. Vaccinated, H1N1 challenged pigs did have significantly less        histopathological changes in their lung tissues compared to        nonvaccinated, H1N1 challenged pigs (p<0.001). These pigs did        not harbor influenza virus antigen in their lung tissues (FIGS.        7 and 8). There was significantly less (p<0.001) infectious        virus present in the lung lavage when compared to the        nonvaccinated H1N1 challenged animals (FIG. 9).

In summary, vaccination of pigs with the attenuated TX/98/del 126 mutantresulted in protective immunity against challenge with a homologousvirus isolate (H3N2 A/Swine/Texas/4199-2/98 virus challenge). Whenvaccinated pigs were challenged with a virus belonging to a differentinfluenza subtype (H1N1 A/Swine/MN/37866/99 virus challenge), thisheterologous virus challenge resulted in significant less lesions inlung tissues and virus load in lung lavage when compared to thenonvaccinated controls at day 5 post inoculation.

These data indicate that attenuated TX/98/del 126 mutant has utility asa modified live virus vaccine showing protective immunity againsthomologous/homotypic challenge and considerable protection againstheterologous/heterotypic challenge.

6.5 Equivalents

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

Various references are cited herein, the disclosure of which areincorporated by reference in their entirety.

What is claimed is:
 1. A genetically engineered attenuated swineinfluenza virus having an impaired interferon antagonist phenotype,wherein the virus comprises a swine influenza virus NS1 gene with amutation resulting in a swine influenza virus NS1 protein having adeletion of between 90 and 94 amino acid residues from thecarboxy-terminus of NS1, wherein the swine influenza virus NS1 gene isfrom A/Swine/Texas/4199-2/98.
 2. The genetically engineered attenuatedswine influenza virus of claim 1, wherein the swine influenza virus isH1N1, H1N2, H3N2, H3N1, H9N2, or H5N1.
 3. The genetically engineeredattenuated swine influenza virus of claim 1, wherein the attenuatedvirus is a reassortant.
 4. The genetically engineered attenuated swineinfluenza virus of claim 1, wherein the attenuated virus is a chimericvirus that expresses a heterologous sequence.
 5. The geneticallyengineered attenuated swine influenza virus of claim 1, wherein theattenuated virus is a chimeric virus that expresses an epitope of aforeign pathogen.
 6. The genetically engineered attenuated swineinfluenza virus of claim 1, wherein the attenuated virus is a chimericvirus that expresses a tumor antigen.
 7. The genetically engineeredattenuated swine influenza virus of claim 1, wherein the attenuatedvirus is engineered to express an epitope from a different virus or atleast one segment derived from a different virus.
 8. The geneticallyengineered attenuated swine influenza virus of claim 7, wherein the atleast one genomic segment is a segment from swine influenza virusselected from the group consisting of: A/Swine/Colorado/1/77,A/Swine/Colorado/23619/99, A/Swine/Cote d'Armor/3633/84, A/Swine/Coted'Armor/3633/84, A/Swine/England/195852/92, A/Swine/Finistere/2899/82,A/Swine/Hong Kong/10/98, A/Swine/Hong Kong/9/98, A/Swine/HongKong/81/78, A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01,A/Swine/Illinois/21587/99, A/Swine/Indiana/1726/88,A/Swine/Indiana/9K035/99, A/Swine/Indiana/P12439/00, A/Swine/Iowa/30,A/Swine/Iowa/15/30, A/Swine/Iowa/533/99, A/Swine/Iowa/569/99,A/Swine/Iowa/3421/90, A/Swine/Iowa/8548-1/98, A/Swine/Iowa/930/01,A/Swine/Iowa/17672/88, A/Swine/Italy/1513-1/98, A/Swine/Italy/1523/98,A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00,A/Swine/Minnesota/593/99, A/Swine/Minnesota/9088-2/98,A/Swine/Nebraska/1/92, A/Swine/Nebraska/209/98,A/Swine/Netherlands/12/85, A/Swine/North Carolina/16497/99,A/Swine/North Carolina/35922/98, A/Swine/North Carolina/93523/01,A/Swine/North Carolina/98225/01, A/Swine/Oedenrode/7C/96,A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/99,A/Swine/Oklahoma/18089/99, A/Swine/Ontario/01911-1/99,A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97,A/Swine/Ontario/97, A/Swine/Quebec/192/81, A/Swine/Quebec/192/91,A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Tennessee/24/77,A/Swine/Texas/4199-2/98, A/Swine/Wisconsin/125/97,A/Swine/Wisconsin/136/97, A/Swine/Wisconsin/163/97,A/Swine/Wisconsin/164/97, A/Swine/Wisconsin/166/97,A/Swine/Wisconsin/168/97, A/Swine/Wisconsin/235/97,A/Swine/Wisconsin/238/97, A/Swine/Wisconsin/457/98,A/Swine/Wisconsin/458/98, A/Swine/Wisconsin/464/98 orA/Swine/Wisconsin/14094/99.
 9. An immunogenic formulation comprising thegenetically engineered attenuated swine influenza virus of claim 1, anda physiologically acceptable excipient.
 10. A pharmaceutical formulationcomprising the genetically engineered attenuated swine influenza virusof claim 1, and a physiologically acceptable excipient.
 11. A culturedcell containing the genetically engineered attenuated swine influenzavirus of claim
 1. 12. The cultured cell of claim 11, wherein the cell isa pig cell or pig cell line.
 13. An embryonated chicken egg containingthe genetically engineered attenuated swine influenza virus of claim 1.14. A genetically engineered attenuated swine influenza virus having animpaired interferon antagonist phenotype, wherein the virus comprises aswine influenza virus NS1 gene with a mutation resulting in a swineinfluenza virus NS1 protein having a deletion of 90 amino acid residuesfrom the carboxy-terminus of NS1, wherein the swine influenza virus NS1gene is from A/Swine/Texas/4199-2/98.
 15. The genetically engineeredattenuated swine influenza virus of claim 14, wherein the swineinfluenza virus is H1N1, H1N2, H3N2, H3N1, H9N2, or H5N1.
 16. Thegenetically engineered attenuated swine influenza virus of claim 14,wherein the attenuated virus is a reassortant.
 17. The geneticallyengineered attenuated swine influenza virus of claim 14, wherein theattenuated virus is a chimeric virus that expresses a heterologoussequence.
 18. The genetically engineered attenuated swine influenzavirus of claim 14, wherein the attenuated virus is a chimeric virus thatexpresses an epitope of a foreign pathogen.
 19. The geneticallyengineered attenuated swine influenza virus of claim 14, wherein theattenuated virus is a chimeric virus that expresses a tumor antigen. 20.The genetically engineered attenuated swine influenza virus of claim 14,wherein the attenuated virus is engineered to express an epitope from adifferent virus or at least one segment derived from a different virus.21. The genetically engineered attenuated swine influenza virus of claim20, wherein the at least one genomic segment is a segment from swineinfluenza virus selected from the group consisting of:A/Swine/Colorado/1/77, A/Swine/Colorado/23619/99, A/Swine/Coted'Armor/3633/84, A/Swine/Cote d'Armor/3633/84,A/Swine/England/195852/92, A/Swine/Finistere/2899/82, A/Swine/HongKong/10/98, A/Swine/Hong Kong/9/98, A/Swine/Hong Kong/81/78,A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01,A/Swine/Illinois/21587/99, A/Swine/Indiana/1726/88,A/Swine/Indiana/9K035/99, A/Swine/Indiana/P12439/00, A/Swine/Iowa/30,A/Swine/Iowa/15/30, A/Swine/Iowa/533/99, A/Swine/Iowa/569/99,A/Swine/Iowa/3421/90, A/Swine/Iowa/8548-1/98, A/Swine/Iowa/930/01,A/Swine/Iowa/17672/88, A/Swine/Italy/1513-1/98, A/Swine/Italy/1523/98,A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00,A/Swine/Minnesota/593/99, A/Swine/Minnesota/9088-2/98,A/Swine/Nebraska/1/92, A/Swine/Nebraska/209/98,A/Swine/Netherlands/12/85, A/Swine/North Carolina/16497/99,A/Swine/North Carolina/35922/98, A/Swine/North Carolina/93523/01,A/Swine/North Carolina/98225/01, A/Swine/Oedenrode/7C/96,A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/99,A/Swine/Oklahoma/18089/99, A/Swine/Ontario/01911-1/99,A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97,A/Swine/Ontario/97, A/Swine/Quebec/192/81, A/Swine/Quebec/192/91,A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Tennessee/24/77,A/Swine/Texas/4199-2/98, A/Swine/Wisconsin/125/97,A/Swine/Wisconsin/136/97, A/Swine/Wisconsin/163/97,A/Swine/Wisconsin/164/97, A/Swine/Wisconsin/166/97,A/Swine/Wisconsin/168/97, A/Swine/Wisconsin/235/97,A/Swine/Wisconsin/238/97, A/Swine/Wisconsin/457/98,A/Swine/Wisconsin/458/98, A/Swine/Wisconsin/464/98 orA/Swine/Wisconsin/14094/99.
 22. An immunogenic formulation comprisingthe genetically engineered attenuated swine influenza virus of claim 14,and a physiologically acceptable excipient.
 23. A pharmaceuticalformulation comprising the genetically engineered attenuated swineinfluenza virus of claim 14, and a physiologically acceptable excipient.24. A cultured cell containing the genetically engineered attenuatedswine influenza virus of claim
 14. 25. The cultured cell of claim 24,wherein the cell is a pig cell or pig cell line.
 26. An embryonatedchicken egg containing the genetically engineered attenuated swineinfluenza virus of claim 14.